Multizymes and their use in making polyunsaturated fatty acids

ABSTRACT

Isolated nucleic acid fragments and recombinant constructs comprising such fragments encoding multizymes (i.e., single polypeptides having at least two independent and separable enzymatic activities) along with a method of making long-chain polyunsaturated fatty acids (PUFAs) using these multizymes in plants and oleaginous yeast are disclosed.

This application claims the benefit of U.S. Provisional Application No.60/909,790, filed Apr. 3, 2007, and U.S. Provisional Application No.61/027,898, filed Feb. 12, 2008, the disclosures of which are herebyincorporated in their entirety.

FIELD OF THE INVENTION

This invention is in the field of biotechnology. More specifically, thisinvention pertains to polynucleotide sequences encoding multizymes andtheir use in the synthesis of long-chain polyunsaturated fatty acids(PUFAs).

BACKGROUND OF THE INVENTION

The importance of PUFAs is undisputed. For example, certain PUFAs areimportant biological components of healthy cells and are recognized as:“essential” fatty acids that cannot be synthesized de novo in mammalsand instead must be obtained either in the diet or derived by furtherelongation and desaturation of linoleic acid (LA; 18:2 omega-6) orα-linolenic acid (ALA; 18:3 omega-3); constituents of plasma membranesof cells, where they may be found in such forms as phospholipids ortriacylglycerols; necessary for proper development (particularly in thedeveloping infant brain) and for tissue formation and repair; and,precursors to several biologically active eicosanoids of importance inmammals (e.g., prostacyclins, eicosanoids, leukotrienes,prostaglandins). Additionally, a high intake of long-chain omega-3 PUFAsproduces cardiovascular protective effects (Dyerberg et al., Amer. J.Clin. Nutr. 28:958-966 (1975); Dyerberg et al., Lancet. 2(8081):117-119(1978); Shimokawa, H., World Rev. Nutr. Diet 88:100-108 (2001); vonSchacky et al., World Rev. Nutr. Diet 88:90-99 (2001)). Numerous otherstudies document wide-ranging health benefits conferred byadministration of omega-3 and/or omega-6 PUFAs against a variety ofsymptoms and diseases (e.g., asthma, psoriasis, eczema, diabetes,cancer).

Today, a variety of different hosts including plants, algae, fungi, andyeast are being investigated as means for commercial PUFA production vianumerous divergent efforts. Although the natural PUFA-producingabilities of the host organisms are sometimes essential to a givenmethodology, genetic engineering has also proven that the naturalabilities of some hosts (even those natively limited to LA and ALA fattyacid production) can be substantially altered to result in high-levelproduction of various long-chain omega-3/omega-6 PUFAs. Whether thiseffect is the result of natural abilities or recombinant technology,arachidonic acid (ARA; 20:4 omega-6), eicosapentaenoic acid (EPA; 20:5omega-3), and docosahexaenoic acid (DHA; 22:6 omega-3) all requireexpression of either the delta-9 elongase/delta-8 desaturase pathway(which operates in some organisms, such as euglenoid species and whichis characterized by the production of eicosadienoic acid (EDA; 20:2omega-6) and/or eicosatrienoic acid (ETrA; 20:3 omega-3)) or the delta-6desaturase/delta-6 elongase pathway (which is predominantly found inalgae, mosses, fungi, nematodes and humans and which is characterized bythe production of gamma-linolenic acid (GLA; 18:3 omega-6) and/orstearidonic acid (STA; 18:4 omega-3)) (FIG. 1). A delta-6 elongase isalso known as a C_(18/20) elongase.

The delta-8 desaturase enzymes identified thus far have the ability toconvert both EDA to dihomo gamma-linolenic acid (DGLA (also known asHGLA); 20:3, n-6) and ETrA to eicosatetraenoic acid (ETA; 20:4, n-3).ARA and EPA are subsequently synthesized from DGLA and ETA,respectively, following reaction with a delta-5 desaturase. DHAsynthesis, however, requires the subsequent expression of an additionalC_(20/22) elongase and a delta-4 desaturase. Most C_(20/22) elongasesidentified so far have the primary ability to convert EPA to DPA, withsecondary activity in converting arachidonic acid (ARA; 20:4 omega-6) todocosatetraenoic acid (DTA; 22:4 omega-6), while most delta-4 desaturaseenzymes identified so far have the primary ability to convert DPA toDHA, with secondary activity in converting docosatetraenoic acid (DTA;22:4 omega-6) to ω-6 docosapentaenoic acid (DPAn-6; 22:5 omega-6).

Based on the role C_(20/22) elongase and delta-4 desaturase enzymes playin the synthesis of DHA, there has been considerable effort to identifyand characterize these enzymes from various sources. As such, numerousC_(20/22) elongases have been disclosed in both the open literature andthe patent literature (e.g., Pavlova sp. CCMP459 (GenBank Accession No.AAV33630), Ostreococcus tauri (GenBank Accession No. AAV67798) andThalassiosira pseudonana (GenBank Accession No. AAV67800)). Similarly,the following delta-4 desaturases have been disclosed: Euglena gracilis(SEQ ID NO:13; GenBank Accession No. AAQ19605; Meyer et al.,Biochemistry, 42(32):9779-9788 (2003)); Thalassiosira pseudonana (SEQ IDNO:29; GenBank Accession No. AAX14506; Tonon et al., FEBS J.,272(13):3401-3412 (2005)); Thraustochytrium aureum (SEQ ID NO:27;GenBank Accession No. AAN75707); Thraustochytrium sp. (GenBank AccessionNo. CAD42496; U.S. Pat. No. 7,087,432); Schizochytrium aggregatum (SEQID NO:28; PCT Publication No. WO 2002/090493); Pavlova lutheri (GenBankAccession No. AAQ98793); and Isochrysis galbana (SEQ ID NO:30; GenBankAccession No. AAV33631; Pereira et al., Biochem. J., 384(2):357-366(2004); PCT Publication No. WO 2002/090493)].

Applicants' Assignee has a number of patent applications concerning theproduction of PUFAs in oleaginous yeasts (i.e., Yarrowia lipolytica),including, for example: U.S. Pat. No. 7,238,482 and No. 7,125,672; U.S.application Ser. No. 11/265,761 (filed Nov. 2, 2005); U.S. applicationSer. No. 11/264,784 (filed Nov. 1, 2005); U.S. application Ser. No.11/264,737 (filed Nov. 1, 2005).

Relatedly, PCT Publication No. WO 2004/071467 (published Aug. 26, 2004)concerns the production of PUFAs in plants, while PCT Publication No. WO2004/071178 (published Aug. 26, 2004) concerns annexin promoters andtheir use in expression of transgenes in plants. Both are Applicants'Assignee's copending applications.

SUMMARY OF THE INVENTION

The present invention concerns a multizyme comprising a singlepolypeptide having at least two independent and separable enzymaticactivities.

In a second embodiment the enzymatic activities of the multizyme can beselected from the group consisting of fatty acid elongases, fatty aciddesaturases, acyl transferases, acyl CoA synthases, and thioesterases.More specifically, the enzymatic activities can comprise at least onefatty acid elongase linked to at least one fatty acid desaturase.

In a third embodiment the multizyme can comprise a first enzymaticactivity linked to a second enzymatic activity and said link is selectedfrom the group consisting of a polypeptide bond, SEQ ID NO:198(EgDHAsyn1 linker), SEQ ID NO:200 (EgDHAsyn2 linker), SEQ ID NO:235(EaDHAsyn1 linker), SEQ ID NO:438, SEQ ID NO:445, SEQ ID NO:472, and SEQID NO:504.

In a fourth embodiment, the invention concerns an isolatedpolynucleotide encoding a DHA synthase comprising:

-   -   (a) a nucleotide sequence encoding a polypeptide having DHA        synthase activity, wherein the polypeptide has at least 80%        amino acid identity, based on the Clustal V method of alignment,        when compared to an amino acid sequence as set forth in SEQ ID        NO:12, SEQ ID NO:22, SEQ ID NO:95, SEQ ID NO:96, or SEQ ID        NO:97;    -   (b) a nucleotide sequence encoding a polypeptide having DHA        synthase activity wherein the nucleotide sequence has at least        80% sequence identity, based on the BLASTN method of alignment,        when compared to a nucleotide sequence as set forth in SEQ ID        NO:11, SEQ ID NO:205, SEQ ID NO:21, SEQ ID NO:91, SEQ ID NO:92,        SEQ ID NO:93, or SEQ ID NO:410;    -   (c) a nucleotide sequence encoding a polypeptide having DHA        synthase activity, wherein the nucleotide sequence hybridizes        under stringent conditions to a nucleotide sequence as set forth        in SEQ ID NO:11, SEQ ID NO:205, SEQ ID NO:21, SEQ ID NO:91, SEQ        ID NO:92, SEQ ID NO:93, or SEQ ID NO:410; or    -   (d) a complement of the nucleotide sequence of (a), (b) or (c),        wherein the complement and the nucleotide sequence consist of        the same number of nucleotides and are 100% complementary.

In a fifth embodiment, the invention concerns the polynucleotideencoding a polypeptide having DHA synthase activity wherein thenucleotide sequence comprises SEQ ID NO:11, SEQ ID NO:21, SEQ ID NO:91,SEQ ID NO:92, SEQ ID NO:93, or SEQ ID NO:410.

In a sixth embodiment, the invention concerns the polypeptide of theinvention having DHA synthase activity, wherein the amino acid sequenceof the polypeptide comprises SEQ ID NO:12, SEQ ID NO:22, SEQ ID NO:95,SEQ ID NO:96, or SEQ ID NO:97.

In a seventh embodiment, the invention concerns an isolatedpolynucleotide encoding a C20 elongase comprising:

-   -   (a) a nucleotide sequence encoding a polypeptide having C20        elongase activity, wherein the polypeptide has at least 80%        amino acid identity, based on the Clustal V method of alignment,        when compared to an amino acid sequence as set forth in SEQ ID        NO:12, SEQ ID NO:22, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97,        SEQ ID NO:202 (EgDHAsyn1 C20 elongase domain), SEQ ID NO:204        (EgDHAsyn2 C20 elongase domain), SEQ ID NO:231 (EaDHAsyn1 C20        elongase domain), SEQ ID NO:232 (EaDHAsyn2 C20 elongase domain)        or SEQ ID NO:233 (EaDHAsyn3 C20 elongase domain);    -   (b) a nucleotide sequence encoding a polypeptide having C20        elongase activity wherein the nucleotide sequence has at least        80% sequence identity, based on the BLASTN method of alignment,        when compared to a nucleotide sequence as set forth in SEQ ID        NO:11, SEQ ID NO:21, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93,        SEQ ID NO:183, SEQ ID NO:188, SEQ ID NO:201 (EgDHAsyn1 C20        elongase domain), SEQ ID NO:206 (EgDHAsyn1* C20 elongase        domain), SEQ ID NO:203 (EgDHAsyn2 C20 elongase domain), SEQ ID        NO:227 (EaDHAsyn1 C20 elongase domain), SEQ ID NO:228 (EaDHAsyn2        C20 elongase domain), SEQ ID NO:229 (EaDHAsyn3 C20 elongase        domain) or SEQ ID NO:230 (EaDHAsyn4 C20 elongase domain);    -   (c) a nucleotide sequence encoding a polypeptide having C20        elongase activity, wherein the nucleotide sequence hybridizes        under stringent conditions to a nucleotide sequence as set forth        in SEQ ID NO:11, SEQ ID NO:21, SEQ ID NO:91, SEQ ID NO:92, SEQ        ID NO:93, SEQ ID NO:183, SEQ ID NO:188, SEQ ID NO:201 (EgDHAsyn1        C20 elongase domain), SEQ ID NO:206 (EgDHAsyn1* C20 elongase        domain), SEQ ID NO:203 (EgDHAsyn2 C20 elongase domain), SEQ ID        NO:227 (EaDHAsyn1 C20 elongase domain), SEQ ID NO:228 (EaDHAsyn2        C20 elongase domain), SEQ ID NO:229 (EaDHAsyn3 C20 elongase        domain) or SEQ ID NO:230 (EaDHAsyn4 C20 elongase domain); or    -   (d) a complement of the nucleotide sequence of (a), (b) or (c),        wherein the complement and the nucleotide sequence consist of        the same number of nucleotides and are 100% complementary.

In an eighth embodiment, the invention concerns an isolatedpolynucleotide encoding a delta-4 desaturase comprising:

-   -   (a) a nucleotide sequence encoding a polypeptide having delta-4        desaturase activity, wherein the polypeptide has at least 80%        amino acid identity, based on the Clustal V method of alignment,        when compared to an amino acid sequence as set forth in SEQ ID        NO:12, SEQ ID NO:22, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97,        SEQ ID NO:193, SEQ ID NO:215, SEQ ID NO:217, SEQ ID NO:221, SEQ        ID NO:239, SEQ ID NO:240, SEQ ID NO:241, SEQ ID NO:246, SEQ ID        NO:247, SEQ ID NO:248, SEQ ID NO:249, SEQ ID NO:382, SEQ ID        NO:384, SEQ ID NO:386, SEQ ID NO:388, SEQ ID NO:404, SEQ ID        NO:406, or SEQ ID NO:408;    -   (b) a nucleotide sequence encoding a polypeptide having delta-4        desaturase activity wherein the nucleotide sequence has at least        80% sequence identity, based on the BLASTN method of alignment,        when compared to a nucleotide sequence as set forth in SEQ ID        NO:11, SEQ ID NO:21, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93,        SEQ ID NO:192, SEQ ID NO:214, SEQ ID No:216, SEQ ID NO:220, SEQ        ID NO:236, SEQ ID NO:237, SEQ ID NO:238, SEQ ID NO:242, SEQ ID        NO:243, SEQ ID NO:244, SEQ ID NO:245, SEQ ID NO:381, SEQ ID        NO:383, SEQ ID NO:385, SEQ ID NO:387, SEQ ID NO:403, SEQ ID        NO:405, or SEQ ID NO:407;    -   (c) a nucleotide sequence encoding a polypeptide having delta-4        desaturase activity, wherein the nucleotide sequence hybridizes        under stringent conditions to a nucleotide sequence as set forth        in SEQ ID NO:11, SEQ ID NO:21, SEQ ID NO:91, SEQ ID NO:92, SEQ        ID NO:93, SEQ ID NO:214, SEQ ID NO:216, SEQ ID NO:220, SEQ ID        NO:236, SEQ ID NO:237, SEQ ID NO:238, SEQ ID NO:242, SEQ ID        NO:243, SEQ ID NO:244, SEQ ID NO:245, SEQ ID NO: 381, SEQ ID        NO:383, SEQ ID NO:385, SEQ ID NO:387, SEQ ID NO:403, SEQ ID        NO:405, or SEQ ID NO:407; or    -   (d) a complement of the nucleotide sequence of (a), (b) or (c),        wherein the complement and the nucleotide sequence consist of        the same number of nucleotides and are 100% complementary.

In a ninth embodiment, the invention concerns an isolated polynucleotideencoding a DHA synthase, said polynucleotide comprising the sequence setforth in any of SEQ ID NO:11, SEQ ID NO:205, SEQ ID NO:21, SEQ ID NO:91,SEQ ID NO:92, SEQ ID NO:93, or SEQ ID NO:410.

In a tenth embodiment, the invention concerns an isolated polynucleotideencoding a C20 elongase, said isolated polynucleotide encoding a C20elongase, said polynucleotide comprising the sequence set forth in anyof SEQ ID NO:183, SEQ ID NO:188, SEQ ID NO:201 (EgDHAsyn1 C20 elongasedomain, SEQ ID NO:206 (EgDHAsyn1* C20 elongase domain), SEQ ID NO:203(EgDHAsyn2 C20 elongase domain), SEQ ID NO:227 (EaDHAsyn1 C20 elongasedomain), SEQ ID NO:228 (EaDHAsyn2 C20 elongase domain), SEQ ID NO:229(EaDHAsyn3 C20 elongase domain) or SEQ ID NO:230 (EaDHAsyn4 C20 elongasedomain).

In an eleventh embodiment, the invention concerns an isolatedpolynucleotide encoding a delta-4 desaturase, said polynucleotidecomprising the sequence set forth in SEQ ID NO:192, SEQ ID NO:214, SEQID NO:220, SEQ ID NO:236, SEQ ID NO:237, SEQ ID NO:238, SEQ ID NO:242,SEQ ID NO:243, SEQ ID NO:244, SEQ ID NO:245, SEQ ID NO:381, SEQ IDNO:383, SEQ ID NO:385, SEQ ID No:387, SEQ ID NO:403, SEQ ID NO:405, orSEQ ID NO:407.

In a twelfth embodiment, the invention concerns a recombinant constructcomprising any of the isolated polynucleotides of the invention operablylinked to at least one regulatory sequence.

In a thirteenth embodiment, the invention concerns a host cellcomprising in its genome the recombinant construct of the invention.More particularly, the host cell is a recombinant microbial host cellcomprising a multizyme of the invention, wherein the first enzymaticactivity is a delta-9 elongase and the second enzymatic activity is adelta-8 desaturase. In another aspect, the first enzymatic activity is aC20 elongase, and the second enzymatic activity is a delta-4 desaturase.

In a fourteenth embodiment, the invention concerns a transformedYarrowia sp. comprising the recombinant construct of the invention.

In a fifteenth embodiment, the invention concerns a method fortransforming a cell, comprising transforming a cell with the recombinantconstruct of the invention and selecting those cells transformed withsaid recombinant construct.

In a sixteenth embodiment, the invention concerns a method for producinga transformed plant comprising transforming a plant cell with any of thepolynucleotides of the invention and regenerating a plant from thetransformed plant cell.

In a seventeenth embodiment, the invention concerns a method forproducing yeast comprising transforming a yeast cell with any of thepolynucleotides of the invention and growing yeast from the transformedyeast cell.

In an eighteenth embodiment, the invention concerns a plant comprisingin its genome the recombinant construct of the invention. Also ofinterest are seeds obtained from such plants, oil obtained from suchseeds, food or feed incorporating such oil, and a beverage incorporatingthe oil of the invention.

In a nineteenth embodiment, the invention concerns an isolated nucleicacid molecule which encodes a C20 elongase as set forth in SEQ ID NO:183wherein at least 147 codons are codon-optimized for expression inYarrowia sp.

In a twentieth embodiment, the invention concerns an isolated nucleicacid molecule which encodes a C20 elongase as set forth in SEQ ID NO:188wherein at least 134 codons are codon-optimized for expression inYarrowia sp.

In a twenty-first embodiment, the invention concerns an isolated nucleicacid molecule which encodes a delta-4 desaturase enzyme as set forth inSEQ ID NO:192 wherein at least 285 codons are codon-optimized forexpression in Yarrowia sp.

In a twenty-second embodiment, the invention concerns a method formaking a multizyme which comprises:

-   -   (a) linking a first polypeptide with at least a second        polypeptide wherein each polypeptide has an independent and        separable enzymatic activity; and    -   (b) evaluating the product of step (a) for the independent and        separable enzymatic activities.

In a twenty-third embodiment, the invention concerns a method foraltering the fatty acid profile of an oilseed plant comprising:

-   -   a) transforming an oilseed plant cell with the recombinant        construct of the invention;    -   b) regenerating a plant from the transformed oilseed plant cell        step (a), wherein the plant has an altered fatty acid profile.

In a twenty-fourth embodiment, the invention concerns an isolatedpolynucleotide encoding a DGLA synthase comprising:

-   -   (a) a nucleotide sequence encoding a polypeptide having DGLA        synthase activity, wherein the polypeptide is set forth in SEQ        ID NO:441, SEQ ID NO:454, SEQ ID NO:461, SEQ ID NO:464, SEQ ID        NO:471, SEQ ID NO:515, SEQ ID NO:516, SEQ ID NO:517, SEQ ID        NO:518, or SEQ ID NO:519;    -   (b) a nucleotide sequence encoding a polypeptide having DGLA        synthase activity wherein the nucleotide sequence is set forth        in SEQ ID NO:440, SEQ ID NO:446, SEQ ID NO:453, SEQ ID NO:460,        SEQ ID NO:463, SEQ ID NO:470, SEQ ID NO:492, SEQ ID NO:493, SEQ        ID NO:494, SEQ ID NO:495, or SEQ ID NO:496;    -   (c) a nucleotide sequence encoding a polypeptide having DGLA        synthase activity, wherein the nucleotide sequence hybridizes        under stringent conditions to a nucleotide sequence as set forth        in SEQ ID NO:440, SEQ ID NO:446, SEQ ID NO:453, SEQ ID NO:460,        SEQ ID NO:463, SEQ ID NO:470, SEQ ID NO:492, SEQ ID NO:493, SEQ        ID NO:494, SEQ ID NO:495, or SEQ ID NO:496; or    -   (d) a complement of the nucleotide sequence of (a), (b) or (c),        wherein the complement and the nucleotide sequence consist of        the same number of nucleotides and are 100% complementary.

In a twenty-fifth embodiment, the invention concerns a method forconverting linoleic acid to dihomo gamma-linolenic acid comprising:

-   -   a) providing a recombinant microbial host cell comprising:        -   i) a DGLA synthase comprising:            -   1) at least one polypeptide encoding a delta-9 elongase;            -   2) at least one polypeptide encoding a delta-8                desaturase; and            -   3) a polypeptide linker;        -   wherein the linker is interposed between the delta-9            elongase and the delta-8 desaturase; and        -   ii) a source of linoleic acid; and    -   b) growing the host cell of (a) under conditions whereby dihomo        gamma-linolenic acid is produced.

In a twenty-sixth embodiment, the invention concerns a method for theconversion of α-linolenic acid to eicosatrienoic acid comprising:

-   -   a) providing a recombinant microbial host cell comprising:        -   i) a DGLA synthase comprising:            -   1) at least one polypeptide encoding a delta-9 elongase;            -   2) at least one polypeptide encoding a delta-8                desaturase; and            -   3) a polypeptide linker;            -   wherein the linker is interposed between the delta-9                elongase and the delta-8 desaturase; and        -   ii) a source of α-linolenic acid; and    -   b) growing the host cell of (a) under conditions whereby        eicosatrienoic acid is produced.

In a twenty-seventh embodiment, the invention concerns a method for theconversion of eicosapentaenoic acid to docosahexaenoic acid comprising:

-   -   a) providing a recombinant microbial host cell comprising:        -   i) a DHA synthase comprising:            -   1) at least one polypeptide encoding a C20 elongase;            -   2) at least one polypeptide encoding a delta-4                desaturase; and            -   3) a polypeptide linker;        -   wherein the linker is interposed between the C20 elongase            and the delta-4 desaturase; and        -   ii) a source of eicosapentaenoic acid; and    -   b) growing the host cell of (a) under conditions whereby        docosahexaenoic acid is produced.

In a twenty-eighth embodiment, the invention concerns a method for theconversion of arachidonic acid to docosapentaenoic acid comprising:

-   -   a) providing a recombinant microbial host cell comprising:        -   i) a DHA synthase comprising:            -   1) at least one polypeptide encoding a C20 elongase;            -   2) at least one polypeptide encoding a delta-4                desaturase; and            -   3) a polypeptide linker;        -   wherein the linker is interposed between the C20 elongase            and the delta-4 desaturase; and        -   ii) a source of arachidonic acid; and    -   b) growing the host cell of (a) under conditions whereby        docosapentaenoic acid is produced.

In a twenty-ninth embodiment, the invention concerns a method for theidentification of a polypeptide having improved delta-4 desaturaseactivity comprising:

-   -   a) providing a wild-type delta-4 desaturase polypeptide isolated        from Euglena anabena having a base-line delta-4 desaturase        activity;    -   b) truncating the wild-type polypeptide of (a) by from about 1        to about 200 amino acids to create a truncated mutant        polypeptide having delta-4 desaturase activity that is increased        as compared with the base-line delta-4 desaturase activity.

In a thirtieth embodiment, the invention concerns a microbial host cellwhich produces a polyunsaturated fatty acid and expresses polypeptidesencoding enzymes in the following sequential pathway:

-   -   1) a delta-9 desaturase,    -   2) a delta-12 desaturase,    -   3) a delta-9 elongase,    -   4) a delta-8 desaturase,    -   5) a delta-5 desaturase,    -   6) a delta-17 desaturase,    -   7) a C_(20/22) elongase, and    -   8) a delta-4 desaturase;

wherein the polypeptides comprise at least one multizyme, a fusioncomprising a fusion between at least one contiguous enzyme pair.

Biological Deposits

The following biological materials have been deposited with the AmericanType Culture Collection (ATCC), 10801 University Boulevard, Manassas,Va. 20110-2209, and bear the following designations, Accession Numbersand dates of deposit (Table 1).

TABLE 1 ATCC Deposit Description Accession Number Date of DepositPlasmid pKR72 ATCC PTA-6019 May 28, 2004 Yarrowia lipolytica Y4128 ATCCPTA-8614 Aug. 23, 2007 Yarrowia lipolytica Y4127 ATCC PTA-8802 Nov. 29,2007

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTINGS

The invention can be more fully understood from the following detaileddescription and the accompanying drawings and Sequence Listing, whichform a part of this application.

FIG. 1 is a representative omega-3 and omega-6 fatty acid pathwayproviding for the conversion of myristic acid through variousintermediates to DHA.

FIG. 2 shows a Clustal W alignment between a portion of the codingsequence of EgDHAsyn2 (SEQ ID NO:21), the cDNA sequence of the Euglenagracilis delta-4 desaturase (SEQ ID NO:23) (NCBI Accession No. AY278558(GI 33466345), locus AY278558, Meyer et al., Biochemistry42(32):9779-9788 (2003)), and the coding sequence of the Euglenagracilis delta-4 desaturase (SEQ ID NO:24) (Meyer et al., supra).

FIGS. 3A and 3B show a Clustal W alignment between the amino acidsequence of EgDHAsyn1 (SEQ ID NO:12), EgDHAsyn2 (SEQ ID NO:22), andEgC20elo1 (SEQ ID NO:6).

FIGS. 4A and 4B show the Clustal W alignment of the N-terminus ofEgDHAsyn1 (SEQ ID NO:12) and the N-terminus of EgDHAsyn2 (SEQ ID NO:22)with EgC20elo1 (SEQ ID NO:6), Pavlova sp. CCMP459 C20-PUFA Elo (SEQ IDNO:2), Ostreococcus tauri PUFA elongase 2 (SEQ ID NO:25) (NCBI AccessionNo. AAV67798 (GI 55852396), locus AAV67798, CDS AY591336; Meyer et al.,J. Lipid Res. 45(10):1899-1909 (2004)), and Thalassiosira pseudonanaPUFA elongase 2 (SEQ ID NO:26) (NCBI Accession No. AAV67800 (GI55852441), locus AAV67800, CDS AY591338; Meyer et al., J. Lipid Res.45(10):1899-1909 (2004)).

FIGS. 5A, 5B, 5C and 5D show the Clustal W alignment of the C-terminusof EgDHAsyn1 (EgDHAsyn1_CT; amino acids 253-793 of SEQ ID NO:12; theN-terminus of EgDHAsyn1 is not shown and is indicated by “ . . . ”) andthe C-terminus of EgDHAsyn2 (EgDHAsyn2_CT; amino acids 253-793 of SEQ IDNO:22, the N-terminus of EgDHAsyn2 is not shown and is indicated by “.”)with Euglena gracilis delta-4 fatty acid desaturase (SEQ ID NO:13),Thraustochytrium aureum delta-4 desaturase (SEQ ID NO:27) (NCBIAccession No. AAN75707 (GI 25956288), locus AAN75707, CDS AF391543),Schizochytrium aggregatum delta-4 desaturase (SEQ ID NO:28) (PCTPublication No. WO 2002/090493), Thalassiosira pseudonana delta-4desaturase (SEQ ID NO:29) (NCBI Accession No. AAX14506 (GI 60173017),locus AAX14506, CDS AY817156; Tonon et al., FEBS J. 272 (13):3401-3412(2005)), and Isochrysis galbana delta-4 desaturase (SEQ ID NO:30) (NCBIAccession No. AAV33631 (GI 54307110), locus AAV33631, CDS AY630574;Pereira et al., Biochem. J., 384(2):357-366 (2004) and PCT PublicationNo. WO 2002/090493).

FIG. 6 shows an alignment of interior fragments of EgDHAsyn1 (EgDHAsyn1_NCT; amino acids 253-365 of SEQ ID NO:12) and EgDHAsyn2 (EgDHAsyn2_NCT;amino acids 253-365 of SEQ ID NO:22) spanning both the C20 elongaseregion and the delta-4 desaturase domain (based on homology) with theC-termini of C20 elongases (EgC20elo1_CT, amino acids 246-298 of SEQ IDNO:6; PavC20elo_CT, amino acids 240-277 of SEQ ID NO:2; OtPUFAelo2_CT,amino acids 256-300 of SEQ ID NO:25; TpPUFAelo2_CT, amino acids 279-358of SEQ ID NO:26) and the N-termini of delta-4 desaturases (EgD4_NT,amino acids 1-116 of SEQ ID NO:13; TaD4_NT, amino acids 1-47 of SEQ IDNO:27; SaD4_NT, amino acids 1-47 of SEQ ID NO:28; TpD4_NT, amino acids1-82 of SEQ ID NO:29; IgD4_NT, amino acids 1-43 of SEQ ID NO:30).

FIG. 7 provides plasmid maps for the following: (A) pY115 (see also SEQID NO:33); (B) Yarrowia lipolytica Gateway® destination vector pBY1 (seealso SEQ ID NO:34); (C) Yarrowia lipolytica Gateway® destination vectorpY159 (see also SEQ ID NO:38); and (D) pBY-EgC20elo1 (see also SEQ IDNO:39).

FIG. 8 provides plasmid maps for the following: (A) pY132 (see also SEQID NO:40); (B) pY161 (see also SEQ ID NO:41); (C) pY164 (see also SEQ IDNO:42); and (D) pY141 (see also SEQ ID NO:49).

FIG. 9 provides plasmid maps for the following: (A) pY143 (see also SEQID NO:52); (B) pY149 (see also SEQ ID NO:55); (C) pY150 (see also SEQ IDNO:62); and (D) pY156 (see also SEQ ID NO:64).

FIG. 10 provides plasmid maps for the following: (A) pY152 (see also SEQID NO:67); (B) pY157 (see also SEQ ID NO:69); (C) pY153 (see also SEQ IDNO:72); and (D) pY151 (see also SEQ ID NO:76).

FIG. 11 is a map of pY160 (see also SEQ ID NO:77).

FIG. 12 shows a chromatogram of the lipid profile of a Euglena anabaenacell extract as described in the Examples.

FIGS. 13A, 13B and 13C show a Clustal W alignment of the amino acidsequences for EaDHAsyn1 (SEQ ID NO:95), EaDHAsyn2 (SEQ ID NO:96),EaDHAsyn3 (SEQ ID NO:97), and EaDHAsyn4 (SEQ ID NO:98).

FIG. 14 provides plasmid maps for the following: (A) pY165 (see also SEQID NO:99); (B) pY166 (see also SEQ ID NO:100); (C) pY167 (see also SEQID NO:101); and (D) pY168 (see also SEQ ID NO:102).

FIG. 15 provides plasmid maps for the following: (A) pKR1061 (see alsoSEQ ID NO:111); (B) pKR973 (see also SEQ ID NO:128); (C) pKR1064 (seealso SEQ ID NO:132); and (D) pKR1133 (see also SEQ ID NO:145).

FIG. 16 provides plasmid maps for the following: (A) pKR1105 (see alsoSEQ ID NO:156); (B) pKR1134 (see also SEQ ID NO:161); (C) pKR1095 (seealso SEQ ID NO:167); and (D) pKR1132 (see also SEQ ID NO:170.

FIG. 17 is a map of KS373 (see also SEQ ID NO:179).

FIG. 18 shows the fatty acid profiles, calculated % elongation, andcalculated % desaturation for the clones (except pBY-EgC20elo1) shown inTable 24.

FIG. 19 shows the fatty acid profiles, calculated % elongation, andcalculated % desaturation for feeding EPA to a vector only control,pY141, pY143, pY149, pY156, pY157, and pY160.

FIG. 20 shows the fatty acid profiles, calculated % elongation, andcalculated % desaturation for feeding DPA to a vector only control,pY141, pY150, pY151, pY152, pY153, pY156, pY157, and pY160.

FIG. 21 shows a schematic of the relative domain structure for eachconstruct described in Table 25.

FIG. 22 shows the fatty acid profiles, calculated % elongation, andcalculated % desaturation for feeding EPA, ARA, and DPA to Yarrowiacells transformed with pY141 (EgDHAsyn1; SEQ ID NO:49) and to a vectoronly control.

FIG. 23 shows the fatty acid profiles for individual embryos from arepresentative event in somatic soybean embryos transformed with soybeanexpression vectors pKR973 and pKR1064 (see Table 26).

FIG. 24 shows the fatty acid profiles from the five best elongationevents in soybean embryos transformed with soybean expression vectorKS373.

FIG. 25 summarizes BLASTP and percent identity values for EgC20elo1(Example 3), EgDHAsyn1 (Example 4), and EgDHAsyn2 (Example 5).

FIG. 26 shows the fatty acid profiles from feeding soybean embryos withEPA. The soybean embryos were selected from the best C20/delta-5elongase and delta-4 desaturase activities in soybean embryostransformed with soybean expression vector pKR1105.

FIG. 27 shows a chromatogram of the lipid profile of a Euglena graciliscell extract as described in the Examples.

FIG. 28 is a map of pKR1183 (see also SEQ ID NO:266).

FIG. 29 summarizes the Euglena anabaena DHA synthase domain sequences.

FIG. 30 is a map of pKR1253 (see also SEQ ID NO:270).

FIG. 31 is a map of pKR1255 (see also SEQ ID NO:275).

FIG. 32 is a map of pKR1189 (see also SEQ ID NO:285).

FIG. 33 is a map of pKR1229 (see also SEQ ID NO:296).

FIG. 34 is a map of pKR1249 (see also SEQ ID NO:297).

FIG. 35 is a map of pKR1322 (see also SEQ ID NO:314).

FIG. 36 shows the fatty acid profiles for five events transformed withpKR1189 that have the lowest average ALA content (average of 5 soybeansomatic embryos analyzed) along with an event (2148-3-8-1) having afatty acid profile typical of wild type embryos for this experiment.Fatty acids are identified as 16:0 (palmitate), 18:0 (stearic acid),18:1 (oleic acid), LA, and ALA. Fatty acid compositions are expressed asa weight percent (wt. %) of total fatty acids.

FIG. 37 shows the fatty acid profiles for five events transformed withpKR1183 that have the highest average DGLA content (average of 5 soybeansomatic embryos analyzed). Fatty acids are identified as 16:0(palmitate), 18:0 (stearic acid), 18:1 (oleic acid), LA, ALA, EDA, ERA,DGLA, and ETA. Fatty acid compositions are expressed as a weight percent(wt. %) of total fatty acids.

FIG. 38 shows the average fatty acid profiles (Average of 10 soybeansomatic embryos) for 20 events transformed with pKR1249 and pKR1253 thathave the highest ARA. Fatty acids are identified as 16:0 (palmitate),18:0 (stearic acid), 18:1 (oleic acid), LA, ALA, EDA, SCI, DGLA, ARA,ERA, JUN, ETA, and EPA. Fatty acid compositions are expressed as aweight percent (wt. %) of total fatty acids. Fatty acids listed as“others” include: 18:2 (5,9), 18:3 (5,9,12), STA, 20:0, 20:1 (11), 20:2(7,11) or 20:2 (8,11), and DPA.

FIG. 39 shows the actual fatty acid profiles for each soybean somaticembryo from one event (AFS 5416-8-1-1) having an average ARA content of17.0% and an average EPA content of 1.5%. Fatty acids are identified as16:0 (palmitate), 18:0 (stearic acid), 18:1 (oleic acid), LA, ALA, EDA,SCI, DGLA, ARA, ERA, JUN, ETA, and EPA. Fatty acid compositions areexpressed as a weight percent (wt. %) of total fatty acids. Fatty acidslisted as “others” include: 18:2 (5,9), 18:3 (5,9,12), STA, 20:0,20:1(11), 20:2 (7,11) or 20:2 (8,11), and DPA.

FIG. 40 shows the average fatty acid profiles (Average of 9 or 10soybean somatic embryos) for 20 events transformed with pKR1249 andpKR1255 that have the highest ARA. Fatty acids are identified as 16:0(palmitate), 18:0 (stearic acid), 18:1 (oleic acid), LA, ALA, EDA, SCI,DGLA, ARA, ERA, JUN, ETA, and EPA; fatty acid compositions are expressedas a weight percent (wt. %) of total fatty acids. Fatty acids listed as“others” include: 18:2 (5,9), 18:3 (5,9,12), STA, 20:0, 20:1 (11), 20:2(7,11) or 20:2 (8,11), and DPA.

FIG. 41 shows the fatty acid profiles from feeding embryos with EPA. Thesoybean embryos were selected from the events with the best C20/delta-5elongase and delta-4 desaturase activities in soybean embryostransformed with soybean expression vector pKR1134. Fatty acids in FIG.41 are identified as 16:0 (palmitate), 18:0 (stearic acid), 18:1 (oleicacid), LA, ALA, EPA, 22:0 (docosanoic acid), DPA, 24:0 (tetracosanoicacid), DHA, and 24:1 (nevonic acid). Fatty acid compositions listed inFIG. 41 are expressed as a weight percent (wt. %) of total fatty acids.

FIG. 42 shows the fatty acid profiles from feeding soybean embryos withEPA. The soybean embryos were selected from the events with the bestC20/delta-5 elongase and delta-4 desaturase activities from the 20 newevents analyzed for soy transformed with pKR1105. Fatty acids in FIG. 42are identified as 16:0 (palmitate), 18:0 (stearic acid), 18:1 (oleicacid), LA, ALA, EPA, 22:0 (docosanoic acid), DPA, 24:0 (tetracosanoicacid), DHA, and 24:1 (nevonic acid). Fatty acid compositions listed inFIG. 42 are expressed as a weight percent (wt. %) of total fatty acids.

FIG. 43 shows a graph depicting the relative activities of eventstransformed with either pKR1105 (C20 elongase and delta-4 desaturaseexpressed individually) or pKR1134 (C20 elongase and delta-4 desaturaseexpressed as a fusion), when the soybean embryos were fed EPA.

FIG. 44 diagrams the development of Yarrowia lipolytica strain Y4305U3.

FIG. 45 provides plasmid maps for the following: (A) pZKLeuN-29E3 and(B) pY116.

FIG. 46 provides plasmid maps for the following: (A) pKO2UF8289 and (B)pZKSL-555R.

FIG. 47 provides plasmid maps for the following: (A) pZP3-Pa777U and (B)pY117.

FIG. 48 provides plasmid maps for the following: (A) pZP2-2988 and (B)pZKUE3S.

FIG. 49 provides plasmid maps for the following: (A) pZKL2-5U89GC and(B) pZKL1-2SP98C.

FIG. 50 provides plasmid maps for the following: (A) pZKUM and (B)pZKD2-5U89A2.

FIG. 51A diagrams the development of Yarrowia lipolytica strain Y4184U.FIG. 51B provides a plasmid map for pEgC20ES.

FIG. 52 provides plasmid maps for the following: (A) pZUFmEgC20ES and(B) pZKL4-220EA4. FIG. 52C is a schematic drawing showing overlap of the3′ region of the EaC20E domain (SEQ ID NO:231) with the 5′ region of theEaD4 domain (SEQ ID NO:246) within EaDHAsyn1 (SEQ ID NO:95).

FIG. 53A shows an alignment between the N-termini of EaD4S (SEQ IDNO:193), EaD4S-1 (SEQ ID NO:382), EaD4S-2 (SEQ ID NO:384), and EaD4S-3(SEQ ID NO:386). FIG. 53B shows an alignment between the N-termini ofEgD4S (SEQ ID NO:388), EgD4S-1 (SEQ ID NO:404), EgD4S-2 (SEQ ID NO:406),and EgD4S-3 (SEQ ID NO:408).

FIG. 54 provides plasmid maps for the following: (A) pZKLY-G204, (B)pEgC20ES-K, (C) pYNTGUS1-CNP, and (D) pZKLY.

FIG. 55 provides plasmid maps for the following: (A) pZUFmG9G8fu and (B)pZUFmG9A8.

FIG. 56 is a map of pKR1014.

FIG. 57 is a map of pKR1152.

FIG. 58 is a map of pKR1151.

FIG. 59 is a map of pKR1150.

FIG. 60 is a map of pKR1199.

FIG. 61 is a map of pKR1200.

FIG. 62 is a map of pKR1184.

FIG. 63 is a map of pKR1321.

FIG. 64 is a map of pKR1326.

For FIGS. 65-71, fatty acids are identified as 16:0 (palmitate), 18:0(stearic acid), 18:1 (oleic acid), LA, ALA, EDA, ERA, DGLA, and ETA, andfatty acid compositions are expressed as a weight percent (wt. %) oftotal fatty acids. In addition, elongation activity is expressed as %delta-9 elongation of C18 fatty acids (C18% delta-9 elong), calculatedaccording to the following formula: ([product]/[substrate+product])*100.More specifically, the combined percent elongation for LA and ALA isdetermined as: ([DGLA+ETA+EDA+ERA]/[LA+ALA+DGLA+ETA+EDA+ERA])*100. Thecombined percent desaturation for EDA and ERA is shown as “C20% delta-8desat”, determined as: ([DGLA+ETA]/[DGLA+ETA+EDA+ERA])*100, and is alsoreferred to as the overall desaturation.

FIG. 65 shows the fatty acid profiles for the five events transformedwith pKR1014 that have the highest average DGLA content (average of 5soybean somatic embryos analyzed).

FIG. 66 shows the fatty acid profiles for the five events transformedwith pKR1152 that have the highest average DGLA content (average of 5soybean somatic embryos analyzed).

FIG. 67 shows the fatty acid profiles for the five events transformedwith pKR1151 that have the highest average DGLA content (average of 5soybean somatic embryos analyzed).

FIG. 68 shows the fatty acid profiles for the five events transformedwith pKR1150 that have the highest average DGLA content (average of 5soybean somatic embryos analyzed).

FIG. 69 shows the fatty acid profiles for the five events transformedwith pKR1199 that have the highest average DGLA content (average of 5soybean somatic embryos analyzed).

FIG. 70 shows the fatty acid profiles for the five events transformedwith pKR1200 that have the highest average DGLA content (average of 5soybean somatic embryos analyzed).

FIG. 71 shows the fatty acid profiles for the five events transformedwith pKR1184 that have the highest average DGLA content (average of 5soybean somatic embryos analyzed).

FIG. 72 shows a comparison of individually expressed delta-9 elongaseswith delta-8 desaturases versus the equivalent delta-9 elongase-delta-8desaturase fusion. Each data point represents the average % DGLA or %EDA for 5-6 embryos (as a % of total fatty acids) for all eventsanalyzed, and Avg. % DGLA is plotted vs. Avg. % EDA. In (A), EgTpomrepresents EgD9e co-expressed with TpomD8 (pKR1014), and EgTpomfusrepresents the EgD9e/TpomD8 fusion (pKR1199). In (B), EgEa representsEgD9e co-expressed with EaD8 (pKR1152), and EgEafus represents theEgD9e/EaD8 fusion (pKR1200). In (C), EaTpom represents EaD9eco-expressed with TpomD8 (pKR1151), and EaTpomfus represents theEaD9e/TpomD8 fusion (pKR1183). In FIG. (D), EaEa represents EaD9eco-expressed with EaD8 (pKR1150) and EaEafus represents the EaD9e/EaD8fusion (pKR1200).

FIG. 73 shows the fatty acid profiles for the five events transformedwith pKR1322 (Experiment MSE2274) that have the highest average ARA andEPA content (average of the 5 embryos analyzed) Fatty acids areidentified as 16:0 (palmitate), 18:0 (stearic acid), 18:1 (oleic acid),18:2 (5,9), LA, ALA, EDA, ERA, SCI, DGLA, JUN (also called JUP), ETA,ARA and EPA. Fatty acid compositions are expressed as a weight percent(wt. %) of total fatty acids. Elongation activity is expressed as %delta-9 elongation of C18 fatty acids (% Elo), calculated according tothe following formula: ([product]/[substrate+product])*100. Morespecifically, the combined percent elongation for LA and ALA isdetermined as:([DGLA+ETA+EDA+ERA+EPA+ARA]/[LA+ALA+DGLA+ETA+EDA+ERA+EPA+ARA])*100. Thecombined percent delta-8 desaturation for EDA and ERA is shown as “%D8”, determined as: ([DGLA+ETA+EPA+ARA]/[DGLA+ETA+EDA+ERA+EPA+ARA])*100.This is also referred to as the overall % delta-8 desaturation. Thecombined percent delta-5 desaturation for DGLA and ETA is shown as “%D5”, determined as: ([EPA+ARA]/[DGLA+ETA+EPA+ARA])*100. This is alsoreferred to as the overall % delta-5 desaturation.

FIG. 74 shows the fatty acid profiles for the five events transformedwith pKR1326 (Experiment MSE2275) that have the highest average DGLA andETA content (average of the 5 embryos analyzed). Fatty acids areidentified as 16:0 (palmitate), 18:0 (stearic acid), 18:1 (oleic acid),LA, ALA, EDA, ERA, DGLA and DGLA and ETA. Fatty acid compositions areexpressed as a weight percent (wt. %) of total fatty acids. Elongationactivity is expressed as % delta-9 elongation of C18 fatty acids (C18%delta-9 elong), calculated according to the following formula:([product]/[substrate+product])*100. More specifically, the combinedpercent elongation for LA and ALA is determined as:([DGLA+ETA+EDA+ERA]/[LA+ALA+DGLA+ETA+EDA+ERA])*100. The combined percentdesaturation for EDA and ERA is shown as “C20% delta-8 desat”,determined as: ([DGLA+ETA]/[DGLA+ETA+EDA+ERA])*100. This is alsoreferred to as the overall desaturation.

The sequence descriptions summarize the Sequences Listing attachedhereto. The Sequence Listing contains one letter codes for nucleotidesequence characters and the single and three letter codes for aminoacids as defined in the IUPAC-IUB standards described in Nucleic AcidsResearch 13:3021-3030 (1985) and in the Biochemical Journal219(2):345-373 (1984).

SEQ ID NOs:1-519 are primers, ORFs encoding genes, proteins (or portionsthereof), or plasmids, as identified in Table 2.

TABLE 2 Summary Of Nucleic Acid And Protein SEQ ID Numbers Nucleic acidProtein Description and Abbreviation SEQ ID NO. SEQ ID NO. M13Funiversal primer 1 Pavlova sp. CCMP459 C20-polyunsaturated fatty — 2(277 AA) acid elongase (GenBank Accession No. AAV33630) Euglena gracilisclone eeg1c.pk005.p14.f 5′ 3 (608 bp) — sequence Euglena gracilis cloneeeg1c.pk005.p14.f full insert 4 (1327 bp) — sequence Euglena gracilisclone eeg1c.pk005.p14.f coding 5 (897 bp) 6 (298 AA) sequence(“EgC20elo1”) Primer SeqE 7 — Primer SeqW 8 — Euglena gracilis cloneeeg1c.pk016.e6.f 5′ 9 (742 bp) — sequence Euglena gracilis cloneeeg1c.pk016.e6.f full insert 10 (2630 bp) — sequence Euglena gracilisclone eeg1c.pk016.e6.f coding 11 (2382 bp) 12 (793 AA) sequence (DHAsynthase 1 or “EgDHAsyn1”) Euglena gracilis delta-4 fatty aciddesaturase — 13 (541 AA) (GenBank Accession No. AAQ19605) M13rev primer14 — EUGel4-1 primer 15 — EgEloD4Mut-5 primer 16 — oEUGel4-2 primer 17 —EgDHAsyn5′ primer 18 — EgDHAsyn3′ primer 19 — Euglena gracilis cloneeeg1c-1 full insert sequence 20 (2630 bp) — Euglena gracilis cloneeeg1c-1 coding sequence 21 (2382 bp) 22 (793 AA) (DHA synthase 2 or“EgDHAsyn2”) Euglena gracilis delta-4 desaturase cDNA 23 (2569 bp) —sequence (GenBank Accession No. AY278558) Euglena gracilis delta-4desaturase-coding 24 (1626 bp) — sequence (GenBank Accession No.AY278558) Ostreococcus tauri PUFA elongase 2 (GenBank — 25 (300 AA)Accession No. AAV67798) Thalassiosira pseudonana PUFA elongase 2 — 26(358 AA) (GenBank Accession No. AAV67800) Thraustochytrium aureumdelta-4 desaturase — 27 (515 AA) (GenBank Accession No. AAN75707)Schizochytrium aggregatum delta-4 desaturase — 28 (509 AA) (PCTPublication No. WO 2002/090493) Thalassiosira pseuduonana delta-4 fattyacid — 29 (550 AA) desaturase (GenBank Accession No. AAX14506)Isochrysis galbana delta-4 desaturase (GenBank — 30 (433 AA) AccessionNo. AAV33631) Plasmid pDMW263 31 (9472 bp) — Plasmid pDMW237 32 (7879bp) — Plasmid pY115 33 (7783 bp) — Plasmid pBY1 34 (8704 bp) — oYFBA1primer 35 — oYFBA1-6 primer 36 — Plasmid pY158 37 (6992 bp) — PlasmidpY159 38 (8707 bp) — Plasmid pBY-C20elo1 39 (8425 bp) — Plasmid pY132 40(9677 bp) — Plasmid pY161 41 (9680 bp) — Plasmid pY164 42 (9701 bp) —EgEPAEloDom-5 primer 43 — oEUG el4-3 primer 44 — Plasmid pKR1062 45(5914 bp) — EgEloD4Mut-5 primer 46 — EgEloD4Mut-3 primer 47 — PlasmidpLF115-7 48 (5914 bp) — Plasmid pY141 49 (9373 bp) — EgDPAEloDom-3primer 50 — Plasmid pHD16 51 (4443 bp) — Plasmid pY143 52 (7903 bp) —oEUGsyn6-2 primer 53 — Plasmid pKR1071 54 (4497 bp) — Plasmid pY149 55(8005 bp) — oEUGsyn6-3 primer 56 — Plasmid pKR1091 57 (4498 bp) —Plasmid pY155 58 (8006 bp) — oRIG6-1 primer 59 — oRIG6-2 primer 60 —Plasmid pKR1067 61 (4827 bp) — Plasmid pY150 62 (8293 bp) — PlasmidpKR1097 63 (5795 bp) — Plasmid pY156 64 (9253 bp) — oEGslne6-1 primer 65— Plasmid pKR1069 66 (4947 bp) — Plasmid pY152 67 (8413 bp) — PlasmidpKR1099 68 (5915 bp) — Plasmid pY157 69 (9373 bp) — oEUGel4-4 primer 70— Plasmid pKR1073 71 (5151 bp) — Plasmid pY153 72 (8617 bp) — oRSA1-1primer 73 — oRSA1-2 primer 74 — Plasmid pKR1068 75 (5058 bp) — PlasmidpY151 76 (8524 bp) — Plasmid pY160 77 (9484 bp) — EaDHAsyn5′ primer 78 —EaDHAsyn5′2 primer 79 — EaDHAsyn5′3 primer 80 — EaDHAsyn5′4 primer 81 —EaDHAsyn3′ primer 82 — EaDHAsyn3′2 primer 83 — EaDHAsyn3′3 primer 84 —EaDHAsyn3′4 primer 85 — EaDHAsyn3′5 primer 86 — Plasmid pLF117-1 87(5582 bp) — Plasmid pLF117-2 88 (5607 bp) — Plasmid pLF117-3 89 (5608bp) — Plasmid pLF117-4 90 (5557 bp) — Euglena anabaena coding sequence(DHA 91 (2523 bp) 95 (841 AA) synthase 1 or “EaDHAsyn1”) Euglenaanabaena coding sequence (DHA 92 (2523 bp) 96 (841 AA) synthase 2 or“EaDHAsyn2”) Euglena anabaena coding sequence (DHA 93 (2523 bp) 97 (841AA) synthase 3 or “EaDHAsyn3”) Euglena anabaena coding sequence (DHA 94(2442 bp) 98 (814 AA) synthase 4 or “EaDHAsyn4”) Plasmid pY165 99(10,133 bp) — Plasmid pY166 100 (10,158 bp) — Plasmid pY167 101 (10,159bp) — Plasmid pY168 102 (10,108 bp) — oEGel2-1 primer 103 — PlasmidpKR1055 104 (5916 bp) — Plasmid pKR72 105 (7085 bp) — oCon-1 primer 106— oCon-2 primer 107 — Plasmid pKR179 108 (4480 bp) — Plasmid pKR1057 109(6873 bp) — Plasmid pKR328 110 (8671 bp) — Plasmid pKR1061 111 (12,844bp) — Euglena gracilis delta-9 elongase (“EgD9elo” or 112 (777 bp) 513(258 AA) “EgD9e” or EgD9E”) oEugEL1-1 primer 113 — oEugEL1-2 primer 114— Plasmid pKR906 115 (4311 bp) — Plasmid pKR132 116 (3983 bp) — PlasmidpKR953 117 (4771 bp) — Plasmid pKR287 118 (5492 bp) — Mortierella alpinadelta-5 desaturase (“MaD5”) 119 (1338 bp) — Plasmid pKR277 120 (2577 bp)— Plasmid pKR952 121 (5364 bp) — Plasmid pKR457 122 (5252 bp) — ModifiedKti/NotI/Kti3′Salb3′ cassette 123 (2635 bp) — Pavlova lutheri delta-8desaturase (“PavD8”) 124 (1269 bp) — PvDES5′Not-1 primer 125 —PvDES3′Not-1 primer 126 — Plasmid pKR970 127 (9276 bp) — Plasmid pKR973128 (11,366 bp) — Plasmid pKR271 129 (6021 bp) — Plasmid pKR226 130(6524 bp) — Plasmid pKR886r 131 (9892 bp) — Plasmid pKR1064 132 (14,066bp) — Plasmid pZBL119 133 (8503 bp) — oSGly-2 primer 134 — oSGly-3primer 135 — Plasmid pPSgly32 136 (3673 bp) — Plasmid pKR142 137 (4509bp) — Plasmid pKR264 138 (4171 bp) — Plasmid pKR1128 139 (6564 bp) —Plasmid pKS129 140 (5671 bp) — Plasmid pKR606 141 (6601 bp) — PlasmidpKR804 142 (6494 bp) — Plasmid pKR1130 143 (10,349 bp) — Plasmid pKR1131144 (4771 bp) — Plasmid pKR1133 145 (12,430 bp) — Plasmid pKS123 146(7048 bp) — oKti5 primer 147 — oKti6 primer 148 — Plasmid pKR124 149(4990 bp) — Plasmid pKR193 150 (4474 bp) — Plasmid pKR1103 151 (5397 bp)— Plasmid pKR1104 152 (9226 bp) — Plasmid pKR300 153 (8649 bp) —Schizochytrium aggregatum delta-4 desaturase* 154 (1607 bp) — (internalAscI site removed) Plasmid pKR1102 155 (6851 bp) — Plasmid pKR1105 156(13,424 bp) — oEUGsyn6-4 primer 157 — Plasmid pKR1107 158 (4498 bp) —Plasmid pKR1112 159 (4461 bp) — Plasmid pKR1115 160 (5989 bp) — PlasmidpKR1134 161 (10,807 bp) — Tetruetreptia pomquetensis CCMP1491 delta-8162 (1260 bp) 514 (420 AA) desaturase (“TpomD8”) TpomNot-5 primer 163 —TpomNot-3 primer 164 — Plasmid pLF114-10 165 (4300 bp) — Plasmid pKR1002166 (5754 bp) — Plasmid pKR1095 167 (11,725 bp) — Plasmid pKR1127 168(5445 bp) — Plasmid pKR1129 169 (9230 bp) — Plasmid pKR1132 170 (11,311bp) — Euglena gracilis DHAsynthase 1 linker 171 (54 bp) — MWG507 primer172 — MWG509 primer 173 — MWG510 primer 174 — MWG511 primer 175 —EgD9elo-EgDHAsyn1Link 176 (839 bp) — Plasmid KS366 177 (5213 bp) —KS366-EgD9elo-EgDHAsyn1Link 178 (5559 bp) — Plasmid KS373 179 (6842 bp)— Conserved motif at C-terminal for C20 elongase — 180 (11 AA) domainsSMART IV primer 181 — Adaptor Primer from the Invitrogen 3′-RACE kit 182— Synthetic C20 elongase derived from Euglena 183 (912 bp) 184 (303 AA)gracilis, codon-optimized for expression in Yarrowia lipolytica(“EgC20ES”) Plasmid pEgC20ES 185 (3632 bp) — NG motif located at theC-terminus of each of the — 186 C20 elongase domains of EgDHAsyn1,EgDHAsyn2 and EgC20elo1 PENGA motif located at the C-terminus of each of— 187 the C20 elongase domains of EgDHAsyn1, EgDHAsyn2 and EgC20elo1Synthetic C20 elongase derived from Euglena 188 (900 bp) 189 (299 AA)anabaena, codon-optimized for expression in Yarrowia lipolytica(“EaC20ES”) Plasmid pEaC20ES 190 (3620 bp) — PCENGTV motif located atthe C-terminus of each 191 of the C20 elongase domains of EgDHAsyn1,EgDHAsyn2 and EgC20elo1 Synthetic delta-4 desaturase derived fromEuglena 192 (1752 bp) 193 (583 AA) anabaena, codon-optimized forexpression in Yarrowia lipolytica (“EaD4S”) Delta-4 desaturase genedomain of EaDHAsyn2 194 (1749 bp) 195 (583 AA) (i.e., corresponding toamino acids 259-841 of SEQ ID NO: 96) Plasmid pEaD4S 196 (4472 bp) —Euglena gracilis EgDHAsyn1 proline-rich linker 197 (54 bp) 198 (18 AA)Euglena gracilis EgDHAsyn2 proline-rich linker 199 (54 bp) 200 (18 AA)Euglena gracilis EgDHAsyn1 C20 elongase domain 201 (909 bp) 202 (303 AA)Euglena gracilis EgDHAsyn2 C20 elongase domain 203 (909 bp) 204 (303 AA)Euglena gracilis EgDHAsyn1* (internal Ncol site 205 (2379 bp) — removed)Euglena gracilis EgDHAsyn1* C20 elongase 206 (909 bp) — domain(“EgDHAsyn1C20EloDom1”) Euglena gracilis EgDHAsyn1* C20 elongase 207(975 bp) 208 (325 AA) domain-proline-rich linker fusion gene(“EgDHAsyn1C20EloDom2Linker”) Isochrysis galbana delta-4 desaturase(“IgD4”) 209 (1299 bp) — Isochrysis galbana delta-4 desaturase (“IgD4*”)210 (1299 bp) 211 (433 AA) (internal SbfI site introduced) In-framefusion between 212 (2259 bp) 213 (753 AA) EgDHAsyn1C20EloDom3Linker andIgD4*, separated by proline-rich linker region(“EgDHAsyn1C20EloDom3-IgD4*”) Euglena gracilis EgDHAsyn1D4Dom1 214 (1416bp) 215 (472 AA) Euglena gracilis EgDHAsyn1D4Dom1* 216 (1419 bp) 217(473 AA) Euglena gracilis EgDHAsyn1C20EloDom3- 218 (2379 bp) 219 (793AA) EgD4Dom1 Euglena gracilis EgDHAsyn1D4Dom2 220 (1623 bp) 221 (541 AA)Schizochytrium aggregatum delta-4 desaturase 222 (1530 bp) — (“SaD4”)Schizochytrium aggregatum delta-4 desaturase 223 (1530 bp) 224 (510 AA)(“SaD4*”) (internal sbfI site introduced) in-frame fusion betweenEgDHAsyn1C20EloDom3 225 (2490 bp) 226 (830 AA) and SaD4*, separated bythe proline-rich linker region (“EgDHAsyn1C20EloDom3-SaD4*”) Euglenaanabaena EaDHAsyn1 C20 elongase 227 (897 bp) 231 (299 AA) domain Euglenaanabaena EaDHAsyn2 C20 elongase 228 (897 bp) 232 (299 AA) domain Euglenaanabaena EaDHAsyn3 C20 elongase 229 (897 bp) 233 (299 AA) domain Euglenaanabaena EaDHAsyn4 C20 elongase 230 (897 bp) — domain Euglena anabaenaEaDHAsyn1 proline-rich linker 234 (99 bp) 235 (33 AA) Euglena anabaenaEaDHAsyn1 delta-4 desaturase 236 (1527 bp) 239 (509 AA) domain 1 Euglenaanabaena EaDHAsyn2 delta-4 desaturase 237 (1527 bp) 240 (509 AA) domain1 Euglena anabaena EaDHAsyn4 delta-4 desaturase 238 (1446 bp) 241 (482AA) domain 1 Euglena anabaena EaDHAsyn1 delta-4 desaturase 242 (1749 bp)246 (583 AA) domain 2 (comprising proline-rich linker and a portion of3′ end of C20 elongase domain) Euglena anabaena EaDHAsyn2 delta-4desaturase 243 (1749 bp) 247 (583 AA) domain 2 (comprising proline-richlinker and a portion of 3′ end of C20 elongase domain) Euglena anabaenaEaDHAsyn3 delta-4 desaturase 244 (1749 bp) 248 (583 AA) domain 2(comprising proline-rich linker and a portion of 3′ end of C20 elongasedomain) Euglena anabaena EaDHAsyn4 delta-4 desaturase 245 (1668 bp) 249(556 AA) domain 2 (comprising proline-rich linker and a portion of 3′end of C20 elongase domain) Plasmid pLF121-1 250 (3668 bp) — PlasmidpLF121-2 251 (3684 bp) — Euglena anabaena delta-9 elongase 1 252 (774bp) 254 (258 AA) (“EaD9Elo1”); also referred to herein as “EaD9E” and“EaD9e” Euglena anabaena delta-9 elongase 2 (“EaD9Elo2”) 253 (774 bp)255 (258 AA) Plasmid pLF119 256 (4276 bp) — Euglena anabaena delta-5desaturase 1 257 (1362 bp) 258 (454 AA) (“EaD5Des1” or “EaD5”) EaD9-5Bbsprimer 259 — EaD9-3fusion primer 260 — EgDHAsyn1Link-5fusion prime 261 —EaD9Elo1-EgDHAsyn1Link 262 (852 bp) — Plasmid pLF124 263 (5559 bp) —Plasmid pKR1177 264 (5559 bp) — Plasmid pKR1179 265 (7916 bp) — PlasmidpKR1183 266 (9190 bp) — Euglena gracilis delta-5 desaturase (“EgD5”) 267(1350 bp) — Plasmid pKR1237 268 (6615 bp) — Plasmid pKR1252 269 (6464bp) — Plasmid pKR1253 270 (10,387 bp) — oEAd5-1-1 primer 271 — oEAd5-1-2primer 272 — Plasmid pKR1136 273 (4899 bp) — Plasmid pKR1139 274 (5592bp) — Plasmid pKR1255 275 (13,293 bp) — Plasmid pKR561 276 (7497 bp) —Soybean delta-15 desaturase (fad3) (GenBank 277 (1143 bp) — AccessionNo. L22964; also called GmFAD3B) HPfad3-1 primer 278 — HPfad3-2 primer279 — HPfad3AB amplicon 280 (709 bp) — HPfad3-3 primer 281 — HPfad3A′-2amplicon 282 (709 bp) — HPfad3ABA′-2 amplicon 283 (1014 bp) — PlasmidpLF129 284 (4526 bp) — Plasmid pKR1189 285 (8503 bp) — Plasmid pKR1209286 (4112 bp) — GmFad3C (GenBank Accession No. AY204712) 287 (1143 bp)288 (380 AA) fad3c-5 primer 289 — fad3c-3 primer 290 — Plasmid pKR1213291 (3764 bp) — Plasmid pKR1218 292 (4353 bp) — Plasmid pKR1210 293(3742 bp) — Plasmid pKR1219 294 (3983 bp) — Plasmid pKR1225 295 (4894bp) — Plasmid pKR1229 296 (8979 bp) — Plasmid pKR1249 297 (10,221 bp) —oEAd9el1-1 primer 298 — oLINK-1 primer 299 — Plasmid pKR1298 300 (4362bp) — oTPd8-1 primer 301 — oTPd8fus-1 primer 302 — oLINK-2 primer 303 —oLINK-1 primer 304 — TpomD8-EgDHAsyn1Link 305 (1335 bp) — PlasmidpKR1291 306 (4848 bp) — Plasmid pKR1301 307 (4284 bp) — Plasmid pKR1301R308 (4284 bp) — Plasmid pKR1311 309 (4228 bp) — Plasmid pKR1304 310(3201 bp) — Plasmid pKR1309 311 (4041 bp) — Plasmid pKR1313 312 (5604bp) — Plasmid pKR1315 313 (6474 bp) — Plasmid pKR1322 314 (10,627 bp) —Plasmid pZKLeuN-29E3 315 (14,688 bp) — Fusarium moniliforme delta-12desaturase 316 (1434 bp) 317 (477 AA) (“FmD12”) Synthetic delta-9elongase derived from Euglena 318 (777 bp) 319 (258 AA) gracilis,codon-optimized for expression in Yarrowia lipolytica (“EgD9eS”)Escherichia coli LoxP recombination site, 320 (34 bp) — recognized by aCre recombinase enzyme Synthetic C_(16/18) elongase derived fromMortierella 321 (828 bp) 322 (275 AA) alpina ELO3, codon-optimized forexpression in Yarrowia lipolytica (“ME3S”) Plasmid pY116 323 (8739 bp) —Plasmid pKO2UF8289 324 (15,337 bp) — Yarrowia lipolytica delta-12desaturase (“YID12”) 325 (1936 bp) 326 (419 AA) Synthetic mutant delta-8desaturase (“EgD8M”; 327 (1272 bp) 328 (422 AA) U.S. patent applicationNo. 11/635258), derived from Euglena gracilis (“EgD8S”; PCT PublicationNo. WO 2006/012326) Euglena gracilis delta-9 elongase (“EgD9e”) 329 (777bp) 330 (258 AA) Plasmid pZKSL-555R 331 (13,707 bp) — Synthetic delta-Sdesaturase derived from Euglena 332 (1350 bp) 333 (449 AA) gracilis(U.S. patent application No. 11/748629), codon-optimized for expressionin Yarrowia lipolytica (“EgD5S”) Synthetic delta-5 desaturase derivedfrom 334 (1392 bp) 335 (463 AA) Peridinium sp. CCMP626 (U.S. patentapplication No. 11/748637), codon-optimized for expression in Yarrowialipolytica (“RD5S”) Euglena gracilis delta-5 desaturase (U.S. patent 336(1350 bp) 337 (449 AA) application No. 11/748629) (“EgD5”) PlasmidpZP3-Pa777U 338 (13,066 bp) — Synthetic delta-17 desaturase derived fromPythium 339 (1080 bp) 340 (359 AA) aphanidermatum, codon-optimized forexpression in Yarrowia lipolytica (U.S. patent application No.11/779915) (“PaD17S”) Pythium aphanidermatum delta-17 desaturase (U.S.341 (1080 bp) 342 (359 AA) patent application No. 11/779915) (“PaD17”)Plasmid pY117 343 (9570 bp) — Yarrowia lipolytica mutantacetohydroxyacid 344 (2987 bp) — synthase (AHAS) gene comprising a W497Lmutation Plasmid pZP2-2988 345 (15,743 bp) — Synthetic delta-12desaturase derived from 346 (1434 bp) 347 (477 AA) Fusarium moniliforme,codon-optimized for expression in Yarrowia lipolytica (“FmD12S”) PlasmidpZKL2-5U89GC 348 (15,812 bp) — Yarrowia lipolytica diacylglycerol 349(1185 bp) 350 (394 AA) cholinephosphotransferase gene (“YICPT1”) PlasmidpZKUE3S 351 (6303 bp) — Plasmid pZKL1-2SP98C 352 (15,877 bp) — PlasmidpZKUM 353 (4313 bp) — Synthetic mutant Ura3 gene comprising a 33 bp 354(1459 bp) — deletion from +21 to +53, a 1 bp deletion at +376 and a 3 bpdeletion from +400 to +403 of the Yarrowia Ura3 coding region (GenBankAccession No. AJ306421) Plasmid pZKD2-5U89A2 355 (15,966 bp) — Yarrowialipolytica diacylglycerol acyltransferase 356 (2119 bp) 357 (514 AA)(DGAT2) (PCT Publication No. WO 2005/003322; U.S. Pat. No. 7,267,976)Synthetic delta-9 elongase derived from Eutreptiella 358 (792 bp) 359(263 AA) sp. CCMP389 codon-optimized for expression in Yarrowialipolytica (“E389D9eS”) Plasmid pZuFmEgC20ES 360 (7904 bp) — PlasmidpZuFmEaC20ES 361 (7892 bp) — Plasmid pZKL4-220EA4 362 (13,412 bp) —Yarrowia lipolytica lipase 4 like locus (GenBank 363 (1221 bp) —Accession No. XM_503825) Plasmid pZuFmEaD4S 364 (8744 bp) — PlasmidpZuFmIgD9ES 365 (7783 bp) — Primer YL921 366 — Primer YL922 367 — PrimerYL923 368 — Primer YL924 369 — Primer YL925 370 — Primer YL926 371 —Plasmid pZuFmEaD4S-M1 372 (8746 bp) — Plasmid pZuFmEaD4S-M2 373 (8747bp) — Plasmid pZuFmEaD4S-M3 374 (8744 bp) — Plasmid pZuFmEaD4S-1 375(8636 bp) — Plasmid pZuFmEaD4S-2 376 (8576 bp) — Plasmid pZuFmEaD4S-3377 (8531 bp) — Plasmid pZKL4-220EA4-1 378 (13,304 bp) — PlasmidpZKL4-220EA4-2 379 (13,244 bp) — Plasmid pZKL4-220EA4-3 380 (13,199 bp)— Truncated synthetic delta-4 desaturase derived 381 (1644 bp) 382 (547AA) from Euglena anabaena, codon-optimized for expression in Yarrowialipolytica (“EaD4S-1”) Truncated synthetic delta-4 desaturase derived383 (1584 bp) 384 (527 AA) from Euglena anabaena, codon-optimized forexpression in Yarrowia lipolytica (“EaD4S-2”) Truncated syntheticdelta-4 desaturase derived 385 (1539 bp) 386 (512 AA) from Euglenaanabaena, codon-optimized for expression in Yarrowia lipolytica(“EaD4S-3”) Synthetic delta-4 desaturase derived from Euglena 387 (1623bp) 388 (540 AA) gracilis, codon-optimized for expression in Yarrowialipolytica (“EgD4S”) Plasmid pEgD4S 389 (4343 bp) — Plasmid pZKL4-220Eg4390 (13,283 bp) — Primer YL935 391 — Primer YL936 392 — Primer YL937 393— Primer YL938 394 — Primer YL939 395 — Primer YL940 396 — PlasmidpEgD4S-M1 397 (4344 bp) — Plasmid pEgD4S-M2 398 (4346 bp) — PlasmidpEgD4S-M3 399 (4346 bp) — Plasmid pZKL4-220Eg4-1 400 (13,202 bp) —Plasmid pZKL4-220Eg4-2 401 (13,133 bp) — Plasmid pZKL4-220Eg4-3 402(13,085 bp) — Truncated synthetic delta-4 desaturase derived 403 (1542bp) 404 (513 AA) from Euglena gracilis, codon-optimized for expressionin Yarrowia lipolytica (“EgD4S-1”) Truncated synthetic delta-4desaturase derived 405 (1473 bp) 406 (490 AA) from Euglena gracilis,codon-optimized for expression in Yarrowia lipolytica (“EgD4S-2”)Truncated synthetic delta-4 desaturase derived 407 (1425 bp) 408 (474AA) from Euglena gracilis, codon-optimized for expression in Yarrowialipolytica (“EgD4S-3”) Plasmid pZKLY-G204 409 (10,417 bp) — SyntheticDHA synthase derived from Euglena 410 (2382 bp) 411 (793 AA) gracilis,codon-optimized for expression in Yarrowia lipolytica (“EgDHAsyn1S”)Plasmid pEgC20ES-K 412 (3632 bp) — Primer YL973 413 — Primer YL974 414 —Plasmid pYNTGUS1-CNP 415 (6652 bp) — Plasmid pZKLY 416 (9045 bp) —Yarrowia lipolytica lipase 7 locus (GenBank 417 (2173 bp) 418 (366 AA)Accession No. AJ549519) Eutreptiella sp. CCMP389 delta-9 elongase 419(792 bp) 420 (263 AA) (“E389D9e”) Synthetic delta-9 elongase derivedfrom Euglena 421 (774 bp) 422 (258 AA) anabaena UTEX 373 codon-optimizedfor expression in Yarrowia lipolytica (“EaD9eS”) Euglena gracilisdelta-8 desaturase (“Eg5” or 423 (1271 bp) 424 (421 AA) “EgD8”)Synthetic delta-8 desaturase derived from Euglena 425 (1272 bp) 426 (422AA) gracilis, codon-optimized for expression in Yarrowia lipolytica(“D8SF” or “EgD8S”) Euglena anabaena UTEX 373 delta-8 desaturase 427(1260 bp) 428 (420 AA) (“EaD8Des3”); also referred to herein as “EaD8”Synthetic delta-8 desaturase derived from Euglena 429 (1260 bp) 430 (420AA) anabaena UTEX 373, codon-optimized for expression in Yarrowialipolytica (“EaD8S”) Plasmid pZuFmEgD9ES 431 (7769 bp) — PlasmidpZuFmEgD9ES-Na 432 (7778 bp) — Primer YL989 433 — Primer YL990 434 —Primer YL991 435 — Primer YL992 436 — Plasmid pKO2UFm8A 437 (8428 bp) —Modified Yarrowia linker — 438 (19 AA) GPARPAGLPPATYYDSLAV PlasmidpZUFmG9G8fu 439 (9098 bp) — EgD9ES/EgD8M gene fusion 440 (2106 bp) 441(701 AA) Plasmid pZUFmG9G8fu-B 442 (9104 bp) — Primer YL1043 443 —Primer YL1044 444 — Modified Yarrowia linker — 445 (24 AA)GAGPARPAGLPPATYYDSLAVMGS EgD9ES/EgD8M gene fusion 446 (2112 bp) 447 (703AA) Plasmid pEaD8S 448 (3988 bp) — Primer YL1059 449 — Primer YL1060 450— Plasmid pEaD8S-B 451 (3990 bp) — Plasmid pZUFmG9A8 452 (9101 bp) —EgD9ES/EaD8S gene fusion 453 (2109 bp) 454 (702 AA) Plasmid pZUFmEaD9ES455 (7769 bp) — Plasmid pZUFmEaD9ES-Na 456 (7775 bp) — Primer YL1049 457— Primer YL1050 458 — Plasmid pZUFmA9G8 459 (9104 bp) — EaD9ES/EgD8Mgene fusion 460 (2112 bp) 461 (703 AA) Plasmid pZUFmA9A8 462 (9101 bp) —EaD9ES/EaD8S gene fusion 463 (2109 bp) 464 (702 AA) Plasmid pE389S 465(3523 bp) — Plasmid pE389S-Na 466 (3529 bp) — Primer YL1051 467 — PrimerYL1052 468 — Plasmid pZUFmR9G8 469 (9119 bp) — E389D9eS/EgD8M genefusion 470 (2127 bp) 471 (708 AA) EgDHAsyn1 proline-rich linker plus 4amino acids 472 (22 AA) Plasmid pKR1007 473 (6267 bp) Plasmid pKR1014474 (11459 bp) Primer EaD8-5 475 (34 bp) Primer EaD8-3 476 (30 bp)Plasmid pLF120-3 477 (4794 bp) Plasmid pKR1138 478 (6526 bp) PlasmidpKR1152 479 (11707 bp) Primer oEAd9el1-2 480 (26 bp) Plasmid pKR1137 481(4310 bp) Plasmid pKR1140 482 (7872 bp) Plasmid pKR1145 483 (6526 bp)Plasmid pKR1151 484 (11706 bp) Plasmid pKR1150 485 (11706 bp) PlasmidpKR1190 486 (5559 bp) Plasmid pKR1195 487 (6833 bp) Plasmid pKR1199 488(9190 bp) Plasmid pKR1196 489 (6833 bp) Plasmid pKR1200 490 (9190 bp)Plasmid pKR1184 491 (9190 bp) EgD9e/TpomD8 fusion 492 (2103 bp) 515 (700AA) EgD9e/EaD8 fusion 493 (2103 bp) 516 (700 AA) EaD9e/TpomD8 fusion 494(2103 bp) 517 (700 AA) EaD9e/EaD8 fusion 495 (2103 bp) 518 (700 AA)EgD9e/PavD8 fusion 496 (2112 bp) 519 (703 AA) Plasmid pKR1303 497 (3967bp) Plasmid pKR1308 498 (4754 bp) Plasmid pKR393 499 (5250 bp) PlasmidpKR407 500 (4140 bp) Plasmid pKR1018 501 (5414 bp) Plasmid pKR1312 502(5731 bp) Plasmid pKR1321 503 (9198 bp) EaDHAsyn1 proline-rich linkerplus 3 amino acids 504 (36 AA) Primer EaLink1 505 (35 bp) Primer EaLink2506 (35 bp) Primer EaLink3 507 (19 bp) EaD9e-EaDHAsyn1Link 508 (894 bp)Plasmid pKR1305 509 (4405 bp) Plasmid pKR1317 510 (3201 bp) PlasmidpKR1320 511 (5816 bp) Plasmid pKR1326 512 (9241 bp)

DETAILED DESCRIPTION OF THE INVENTION

The disclosure of each reference set forth herein is hereby incorporatedby reference in its entirety.

The present invention relates to multizymes, such as DHA synthase. Theseare useful for, inter alia, the manipulation of biochemical pathways forthe production of healthful PUFAs and more specifically for theproduction of docosahexaenoic acid (DHA). Thus, the subject inventionfinds many applications. PUFAs, or derivatives thereof, made by themethodology disclosed herein can be used as dietary substitutes, orsupplements, particularly infant formulas, for patients undergoingintravenous feeding or for preventing or treating malnutrition.

Alternatively, the purified PUFAs (or derivatives thereof) may beincorporated into cooking oils, fats, or margarines formulated so thatin normal use the recipient would receive the desired amount for dietarysupplementation. The PUFAs may also be incorporated into infantformulas, nutritional supplements, or other food products and may finduse as anti-inflammatory or cholesterol lowering agents. Optionally, thecompositions may be used for pharmaceutical use (human or veterinary).In this case, the PUFAs are generally administered orally but can beadministered by any route by which they may be successfully absorbed,e.g., parenterally (e.g., subcutaneously, intramuscularly orintravenously), rectally, vaginally, or topically (e.g., as a skinointment or lotion).

Supplementation of humans or animals with PUFAs produced by recombinantmeans can result in increased levels of the added PUFAs, as well astheir metabolic progeny. For example, treatment with EPA can result notonly in increased levels of EPA, but also downstream products of EPAsuch as eicosanoids (i.e., prostaglandins, leukotrienes, thromboxanes).Complex regulatory mechanisms can make it desirable to combine variousPUFAs, or add different conjugates of PUFAs, in order to prevent,control, or overcome such mechanisms to achieve the desired levels ofspecific PUFAs in an individual.

DEFINITIONS

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, reference to “a plant” includes aplurality of such plants, reference to “a cell” includes one or morecells and equivalents thereof known to those skilled in the art, and soforth.

The term “invention” or “present invention” as used herein is not meantto be limiting to any one specific embodiment of the invention butapplies generally to any and all embodiments of the invention asdescribed in the claims and specification.

In the context of this disclosure, a number of terms and abbreviationsare used. The following definitions are provided.

“Open reading frame” is abbreviated ORF.

“Polymerase chain reaction” is abbreviated PCR.

“American Type Culture Collection” is abbreviated ATCC.

“Polyunsaturated fatty acid(s)” is abbreviated PUFA(s).

“Triacylglycerols” are abbreviated TAGs.

The terms “down-regulate or down-regulation”, as used herein, refer to areduction or decrease in the level of expression of a gene orpolynucleotide.

The term “multizyme” refers to a single polypeptide having at least twoindependent and separable enzymatic activities. Preferably, themultizyme comprises a first enzymatic activity linked to a secondenzymatic activity.

The term “fusion protein” is used interchangeably with the term“multizyme”. Thus, a “fusion protein” refers to a single polypeptidehaving at least two independent and separable enzymatic activities.

The term “fusion gene” refers to a polynucleotide or gene that encodes amultizyme. A fusion gene can be constructed by linking at least two DNAfragments, wherein each DNA fragment encodes for an independent andseparate enzyme activity. An example of a fusion gene is describedherein below in Example 38, in which the Hybrid1-HGLA Synthase fusiongene was constructed by linking the Euglena anabaena delta-9 elongase(EaD9Elo1; SEQ ID NO:252) and the Tetruetreptia pomquetensis CCMP1491delta-8 desaturase (TpomD8; SEQ ID NO:162) using the Euglena gracilisDHA synthase 1 proline-rich linker. (EgDHAsyn1Link; SEQ ID NO:197).

A “domain” or “functional domain” is a discrete, continuous part orsubsequence of a polypeptide that can be associated with a function(e.g. enzymatic activity). As used herein, the term “domain” includesbut is not limited to fatty acid biosynthetic enzymes and portions offatty acid biosynthetic enzymes that retain enzymatic activity.

“DHA synthase” is an example of a multizyme. Specifically, a DHAsynthase comprises a C20 elongase linked to a delta-4 desaturase usingany of the linkers described herein. Another example of a multizyme is asingle polypeptide comprising a delta-9 elongase linked to a delta-8desaturase as discussed below.

The term “link” refers to joining or bonding at least two polypeptideshaving independent and separable enzyme activities.

The term “linker” refers to the bond or link between two or morepolypeptides each having independent and separable enzymatic activities

The link used to form a multizyme is minimally comprised of a singlepolypeptide bond. In another aspect, the link may be comprised of oneamino acid residue, such as proline, or a polypeptide. If the link is apolypeptide, it may be desirable for the link to have at least oneproline amino acid residue.

An example of a linker is shown in SEQ ID NO:198 (the EgDHAsyn1proline-rich linker).

The term “fatty acids” refers to long-chain aliphatic acids (alkanoicacids) of varying chain lengths, from about C₁₂ to C₂₂ (although bothlonger and shorter chain-length acids are known). The predominant chainlengths are between C₁₆ and C₂₂. Additional details concerning thedifferentiation between “saturated fatty acids” versus “unsaturatedfatty acids”, “monounsaturated fatty acids” versus “polyunsaturatedfatty acids” (or “PUFAs”), and “omega-6 fatty acids” (ω-6 or n-6) versus“omega-3 fatty acids” (omega-3 or n-3) are provided in U.S. Pat. No.7,238,482.

Fatty acids are described herein by a simple notation system of “X:Y”,wherein X is the total number of carbon (C) atoms in the particularfatty acid and Y is the number of double bonds. The number following thefatty acid designation indicates the position of the double bond fromthe carboxyl end of the fatty acid with the “c” affix for thecis-configuration of the double bond (e.g., palmitic acid (16:0),stearic acid (18:0), oleic acid (18:1, 9c), petroselinic acid (18:1,6c), LA (18:2, 9c, 12c), GLA (18:3, 6c, 9c, 12c) and ALA (18:3, 9c, 12c,15c)). Unless otherwise specified, 18:1, 18:2 and 18:3 refer to oleic,LA and ALA fatty acids, respectively. If not specifically written asotherwise, double bonds are assumed to be of the cis configuration. Forinstance, the double bonds in 18:2 (9,12) would be assumed to be in thecis configuration.

Nomenclature used to describe PUFAs in the present disclosure is shownbelow in Table 3. In the column titled “Shorthand Notation”, theomega-reference system is used to indicate the number of carbons, thenumber of double bonds and the position of the double bond closest tothe omega carbon, counting from the omega carbon (which is numbered 1for this purpose). The remainder of the table summarizes the commonnames of omega-3 and omega-6 fatty acids and their precursors, theabbreviations that will be used throughout the remainder of thespecification, and each compounds' chemical name.

TABLE 3 Nomenclature of Polyunsaturated Fatty Acids and PrecursorsCommon Shorthand Name Abbreviation Chemical Name Notation myristic —tetradecanoic 14:0 palmitic PA or hexadecanoic 16:0 Palmitatepalmitoleic — 9-hexadecenoic 16:1 stearic — octadecanoic 18:0 oleic —cis-9-octadecenoic 18:1 linoleic LA cis-9,12-octadecadienoic 18:2 ω-6gamma- GLA cis-6,9,12-octadecatrienoic 18:3 ω-6 linolenic eicosadienoicEDA cis-11,14-eicosadienoic 20:2 ω-6 dihomo- DGLA orcis-8,11,14-eicosatrienoic 20:3 ω-6 gamma- HGLA (used linolenicinterchangeably herein) sciadonic SCI cis-5,11,14-eicosatrienoic 20:3bω-6 arachidonic ARA cis-5,8,11,14- 20:4 ω-6 eicosatetraenoic alpha- ALAcis-9,12,15- 18:3 ω-3 linolenic octadecatrienoic stearidonic STAcis-6,9,12,15- 18:4 ω-3 octadecatetraenoic eicosatrienoic ETrA orcis-11,14,17- 20:3 ω-3 ERA eicosatrienoic eicosa- ETA cis-8,11,14,17-20:4 ω-3 tetraenoic eicosatetraenoic juniperonic JUP cis-5,11,14,17-20:4b ω-3 eicosatrienoic eicosa- EPA cis-5,8,11,14,17- 20:5 ω-3pentaenoic eicosapentaenoic docosa- DRA cis-10,13,16- 22:3 ω-3 trienoicdocosatrienoic docosa- DTA cis-7,10,13,16- 22:4 ω-3 tetraenoicdocosatetraenoic docosa- DPAn-6 cis-4,7,10,13,16- 22:5 ω-6 pentaenoicdocosapentaenoic docosa- DPA cis-7,10,13,16,19- 22:5 ω-3 pentaenoicdocosapentaenoic docosa- DHA cis-4,7,10,13,16,19- 22:6 ω-3 hexaenoicdocosahexaenoic

A metabolic, or biosynthetic, pathway, in a biochemical sense, can beregarded as a series of chemical reactions occurring within a cell,catalyzed by enzymes, to achieve either the formation of a metabolicproduct to be used or stored by the cell, or the initiation of anothermetabolic pathway (then called a flux generating step). Many of thesepathways are elaborate, and involve a step by step modification of theinitial substance to shape it into a product having the exact chemicalstructure desired.

The term “PUFA biosynthetic pathway” refers to a metabolic process thatconverts oleic acid to LA, EDA, GLA, DGLA, ARA, DTA, DPAn-6, ALA, STA,ETrA, ETA, EPA, DPA and DHA. This process is well described in theliterature (e.g., see PCT Publication No. WO 2006/052870).Simplistically, this process involves elongation of the carbon chainthrough the addition of carbon atoms and desaturation of the moleculethrough the addition of double bonds, via a series of specialdesaturation and elongation enzymes (i.e., “PUFA biosynthetic pathwayenzymes”) present in the endoplasmic reticulum membrane. Morespecifically, “PUFA biosynthetic pathway enzyme” refers to any of thefollowing enzymes (and genes which encode said enzymes) associated withthe biosynthesis of a PUFA, including: a delta-4 desaturase, a delta-5desaturase, a delta-6 desaturase, a delta-12 desaturase, a delta-15desaturase, a delta-17 desaturase, a delta-9 desaturase, a delta-8desaturase, a delta-9 elongase, a C_(14/16) elongase, a C_(16/18)elongase, a C_(18/20) elongase, a C_(20/22) elongase, a DHA synthaseand/or a multizyme of the instant invention.

The term “omega-3/omega-6 fatty acid biosynthetic pathway” refers to aset of genes which, when expressed under the appropriate conditionsencode enzymes that catalyze the production of either or both omega-3and omega-6 fatty acids. Typically the genes involved in theomega-3/omega-6 fatty acid biosynthetic pathway encode PUFA biosyntheticpathway enzymes. A representative pathway is illustrated in FIG. 1,providing for the conversion of myristic acid through variousintermediates to DHA, which demonstrates how both omega-3 and omega-6fatty acids may be produced from a common source. The pathway isnaturally divided into two portions where one portion will generateomega-3 fatty acids and the other portion, omega-6 fatty acids.

The term “functional” as used herein in context with the omega-3/omega-6fatty acid biosynthetic pathway means that some (or all) of the genes inthe pathway express active enzymes, resulting in in vivo catalysis orsubstrate conversion. It should be understood that “omega-3/omega-6fatty acid biosynthetic pathway” or “functional omega-3/omega-6 fattyacid biosynthetic pathway” does not imply that all the PUFA biosyntheticpathway enzyme genes are required, as a number of fatty acid productswill only require the expression of a subset of the genes of thispathway.

The term “delta-6 desaturase/delta-6 elongase pathway” refers to a PUFAbiosynthetic pathway that minimally includes at least one delta-6desaturase and at least one C_(18/20) elongase, thereby enablingbiosynthesis of DGLA and/or ETA from LA and ALA, respectively, with GLAand/or STA as intermediate fatty acids. With expression of otherdesaturases and elongases, ARA, DTA, DPAn-6, EPA, DPA, and DHA may alsobe synthesized.

The term “delta-9 elongase/delta-8 desaturase pathway” refers to a PUFAbiosynthetic pathway that minimally comprises at least one delta-9elongase and at least one delta-8 desaturase, thereby enablingbiosynthesis of DGLA and/or ETA from LA and ALA, respectively, with EDAand/or ETrA as intermediate fatty acids With expression of otherdesaturases and elongases, ARA, DTA, DPAn-6, EPA, DPA and DHA may alsobe synthesized. This pathway may be advantageous in some embodiments, asthe biosynthesis of GLA and/or STA is excluded.

The term “intermediate fatty acid” refers to any fatty acid produced ina fatty acid metabolic pathway that can be further converted to anintended product fatty acid in this pathway by the action of othermetabolic pathway enzymes. For instance, when EPA is produced using thedelta-9 elongase/delta-8 desaturase pathway, EDA, ETrA, DGLA, ETA andARA can be produced and are considered “intermediate fatty acids” sincethese fatty acids can be further converted to EPA via action of othermetabolic pathway enzymes.

The term “by-product fatty acid” refers to any fatty acid produced in afatty acid metabolic pathway that is not the intended fatty acid productof the pathway nor an “intermediate fatty acid” of the pathway. Forinstance, when EPA is produced using the delta-9 elongase/delta-8desaturase pathway, sciadonic acid (SCI) and juniperonic acid (JUP) alsocan be produced by the action of a delta-5 desaturase on either EDA orETrA, respectively. They are considered to be “by-product fatty acids”since neither can be further converted to EPA by the action of othermetabolic pathway enzymes.

The terms “triacylglycerol”, “oil” and “TAGs” refer to neutral lipidscomposed of three fatty acyl residues esterified to a glycerol molecule(and such terms will be used interchangeably throughout the presentdisclosure herein). Such oils can contain long-chain PUFAs, as well asshorter saturated and unsaturated fatty acids and longer chain saturatedfatty acids. Thus, “oil biosynthesis” generically refers to thesynthesis of TAGs in the cell.

“Percent (%) PUFAs in the total lipid and oil fractions” refers to thepercent of PUFAs relative to the total fatty acids in those fractions.The term “total lipid fraction” or “lipid fraction” both refer to thesum of all lipids (i.e., neutral and polar) within an oleaginousorganism, thus including those lipids that are located in thephosphatidylcholine (PC) fraction, phosphatidylethanolamine (PE)fraction and triacylglycerol (TAG or oil) fraction. However, the terms“lipid” and “oil” will be used interchangeably throughout thespecification.

The terms “conversion efficiency” and “percent substrate conversion”refer to the efficiency by which a particular enzyme (e.g., adesaturase) can convert substrate to product. The conversion efficiencyis measured according to the following formula:([product]/[substrate+product])*100, where ‘product’ includes theimmediate product and all products in the pathway derived from it.

“Desaturase” is a polypeptide that can desaturate, i.e., introduce adouble bond, in one or more fatty acids to produce a fatty acid orprecursor of interest. Despite use of the omega-reference systemthroughout the specification to refer to specific fatty acids, it ismore convenient to indicate the activity of a desaturase by countingfrom the carboxyl end of the substrate using the delta-system. Forexample, delta-8 desaturases will desaturate a fatty acid between theeighth and ninth carbon atom numbered from the carboxyl-terminal end ofthe molecule and can, for example, catalyze the conversion of EDA toDGLA and/or ETrA to ETA. Other useful fatty acid desaturases include,for example: (1) delta-5 desaturases that catalyze the conversion ofDGLA to ARA and/or ETA to EPA; (2) delta-6 desaturases that catalyze theconversion of LA to GLA and/or ALA to STA; (3) delta-4 desaturases thatcatalyze the conversion of DPA to DHA and/or DTA to DPAn-6; (4) delta-12desaturases that catalyze the conversion of oleic acid to LA; (5)delta-15 desaturases that catalyze the conversion of LA to ALA and/orGLA to STA; (6) delta-17 desaturases that catalyze the conversion of ARAto EPA and/or DGLA to ETA; and (7) delta-9 desaturases that catalyze theconversion of palmitic acid to palmitoleic acid (16:1) and/or stearicacid to oleic acid (18:1). In the art, delta-15 and delta-17 desaturasesare also occasionally referred to as “omega-3 desaturases”, “w-3desaturases”, and/or “n-3 desaturases”, based on their ability toconvert omega-6 fatty acids into their omega-3 counterparts (e.g.,conversion of LA into ALA and ARA into EPA, respectively). In someembodiments, it is most desirable to empirically determine thespecificity of a particular fatty acid desaturase by transforming asuitable host with the gene for the fatty acid desaturase anddetermining its effect on the fatty acid profile of the host.

The term “delta-4 desaturase” refers to an enzyme that will desaturate afatty acid between the fourth and fifth carbon atom numbered from thecarboxyl-terminal end of the molecule and that can, for example,catalyze the conversion of DPA to DHA and/or DTA to DPAn-6. For thepurposes herein, the term “EgDHAsyn1” refers to a DHA synthase enzyme(SEQ ID NO:12) isolated from Euglena gracilis, encoded by SEQ ID NO:11herein. The term “EgDHAsyn2” refers to a DHA synthase enzyme (SEQ IDNO:22) isolated from Euglena gracilis, encoded by SEQ ID NO:21 herein.The term “EaDHAsyn1” refers to a DHA synthase enzyme (SEQ ID NO:95)isolated from Euglena anabaena, encoded by SEQ ID NO:91 herein. The term“EaDHAsyn2” refers to a DHA synthase enzyme (SEQ ID NO:96) isolated fromEuglena anabaena, encoded by SEQ ID NO:92 herein. The term “EaDHAsyn3”refers to a DHA synthase enzyme (SEQ ID NO:97) isolated from Euglenaanabaena, encoded by SEQ ID NO:93 herein. The term “EaDHAsyn4” refers toan enzyme (SEQ ID NO:98) isolated from Euglena anabaena, encoded by SEQID NO:94 herein.

The term “elongase system” refers to a suite of four enzymes that areresponsible for elongation of a fatty acid carbon chain to produce afatty acid that is two carbons longer than the fatty acid substrate thatthe elongase system acts upon. More specifically, the process ofelongation occurs in association with fatty acid synthase, whereby CoAis the acyl carrier (Lassner et al., Plant Cell 8:281-292 (1996)). Inthe first step, which has been found to be both substrate-specific andalso rate-limiting, malonyl-CoA is condensed with a long-chain acyl-CoAto yield carbon dioxide (CO₂) and a β-ketoacyl-CoA (where the acylmoiety has been elongated by two carbon atoms). Subsequent reactionsinclude reduction to β-hydroxyacyl-CoA, dehydration to an enoyl-CoA anda second reduction to yield the elongated acyl-CoA. Examples ofreactions catalyzed by elongase systems are the conversion of GLA toDGLA, STA to ETA, LA to EDA, ALA to ETrA and EPA to DPA.

For the purposes herein, an enzyme catalyzing the first condensationreaction (i.e., conversion of malonyl-CoA and long-chain acyl-CoA toβ-ketoacyl-CoA) will be referred to generically as an “elongase”. Ingeneral, the substrate selectivity of elongases is somewhat broad butsegregated by both chain length and the degree of unsaturation.Accordingly, elongases can have different specificities. For example, aC_(14/16) elongase will utilize a C₁₄ substrate (e.g., myristic acid); aC_(16/18) elongase will utilize a C₁₆ substrate (e.g., palmitate); aC_(18/20) elongase will utilize a C₁₈ substrate (e.g., GLA, STA); and aC_(20/22) elongase will utilize a C₂₀ substrate (e.g., ARA, EPA).Similarly, a “delta-9 elongase” is able to catalyze the conversion of LAto EDA and/or ALA to ETrA.

It is important to note that some elongases have broad specificity andthus a single enzyme may be capable of catalyzing several elongasereactions. Thus, for example, a delta-9 elongase may also act as aC_(16/18) elongase, C_(18/20) elongase and/or C_(20/22) elongase and mayhave alternate, but not preferred, specificities for delta-5 and delta-6fatty acids such as EPA and/or GLA, respectively.

The term “C20 elongase” as used herein refers to an enzyme whichutilizes a C20 substrate such as EPA or ARA, for example. The term“C20/delta-5 elongase” refers to an enzyme that utilizes a C20 substratewith a delta-5 double bond.

Similarly for the purposes herein, the term “EgD9elo” or “EgD9e” refersto a delta-9 elongase isolated from Euglena gracilis (see SEQ ID NO:112;also see U.S. application Ser. No. 11/601,563 (filed Nov. 16, 2006,which published as US-2007-0118929-A1 on May 24, 2007)).

As used herein, “nucleic acid” means a polynucleotide and includes asingle or double-stranded polymer of deoxyribonucleotide orribonucleotide bases. Nucleic acids may also include fragments andmodified nucleotides. Thus, the terms “polynucleotide”, “nucleic acidsequence”, “nucleotide sequence” or “nucleic acid fragment” are usedinterchangeably and refer to a polymer of RNA or DNA that is single- ordouble-stranded, optionally containing synthetic, non-natural or alterednucleotide bases. Nucleotides (usually found in their 5′-monophosphateform) are referred to by their single letter designation as follows: “A”for adenylate or deoxyadenylate (for RNA or DNA, respectively), “C” forcytidylate or deoxycytidylate, “G” for guanylate or deoxyguanylate, “U”for uridylate, “T” for deoxythymidylate, “R” for purines (A or G), “Y”for pyrimidines (C or T), “K” for G or T, “H” for A or C or T, “I” forinosine, and “N” for any nucleotide.

The terms “subfragment that is functionally equivalent” and“functionally equivalent subfragment” are used interchangeably herein.These terms refer to a portion or subsequence of an isolated nucleicacid fragment in which the ability to alter gene expression or produce acertain phenotype is retained whether or not the fragment or subfragmentencodes an active enzyme. For example, the fragment or subfragment canbe used in the design of chimeric genes to produce the desired phenotypein a transformed plant. Chimeric genes can be designed for use insuppression by linking a nucleic acid fragment or subfragment thereof,whether or not it encodes an active enzyme, in the sense or antisenseorientation relative to a plant promoter sequence.

The term “conserved domain” or “motif” means a set of amino acidsconserved at specific positions along an aligned sequence ofevolutionarily related proteins. While amino acids at other positionscan vary between homologous proteins, amino acids that are highlyconserved at specific positions indicate amino acids that are essentialin the structure, the stability, or the activity of a protein. The terms“homology”, “homologous”, “substantially similar” and “correspondingsubstantially” are used interchangeably herein. They refer to nucleicacid fragments wherein changes in one or more nucleotide bases do notaffect the ability of the nucleic acid fragment to mediate geneexpression or produce a certain phenotype. These terms also refer tomodifications of the nucleic acid fragments of the instant inventionsuch as deletion or insertion of one or more nucleotides that do notsubstantially alter the functional properties of the resulting nucleicacid fragment relative to the initial, unmodified fragment. It istherefore understood, as those skilled in the art will appreciate, thatthe invention encompasses more than the specific exemplary sequences.

Moreover, the skilled artisan recognizes that substantially similarnucleic acid sequences encompassed by this invention are also defined bytheir ability to hybridize (under moderately stringent conditions, e.g.,0.5×SSC, 0.1% SDS, 60° C.) with the sequences exemplified herein, or toany portion of the nucleotide sequences disclosed herein and which arefunctionally equivalent to any of the nucleic acid sequences disclosedherein. Stringency conditions can be adjusted to screen for moderatelysimilar fragments, such as homologous sequences from distantly relatedorganisms, to highly similar fragments, such as genes that duplicatefunctional enzymes from closely related organisms. Post-hybridizationwashes determine stringency conditions.

The term “selectively hybridizes” includes reference to hybridization,under stringent hybridization conditions, of a nucleic acid sequence toa specified nucleic acid target sequence to a detectably greater degree(e.g., at least 2-fold over background) than its hybridization tonon-target nucleic acid sequences and to the substantial exclusion ofnon-target nucleic acids. Selectively hybridizing sequences typicallyhave about at least 80% sequence identity, or 90% sequence identity, upto and including 100% sequence identity (i.e., fully complementary) witheach other.

The term “stringent conditions” or “stringent hybridization conditions”includes reference to conditions under which a probe will selectivelyhybridize to its target sequence. Stringent conditions aresequence-dependent and will be different in different circumstances. Bycontrolling the stringency of the hybridization and/or washingconditions, target sequences can be identified which are 100%complementary to the probe (homologous probing). Alternatively,stringency conditions can be adjusted to allow some mismatching insequences so that lower degrees of similarity are detected (heterologousprobing). Generally, a probe is less than about 1000 nucleotides inlength, optionally less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37° C., and awash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the T_(m) can be approximated fromthe equation of Meinkoth et al., Anal. Biochem. 138:267-284 (1984):T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M isthe molarity of monovalent cations, % GC is the percentage of guanosineand cytosine nucleotides in the DNA, % form is the percentage offormamide in the hybridization solution, and L is the length of thehybrid in base pairs. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of a complementary target sequencehybridizes to a perfectly matched probe. T_(m) is reduced by about 1° C.for each 1% of mismatching; thus, T_(m), hybridization and/or washconditions can be adjusted to hybridize to sequences of the desiredidentity. For example, if sequences with ≧90% identity are sought, theT_(m) can be decreased 10° C. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence and its complement at a defined ionic strengthand pH. However, severely stringent conditions can utilize ahybridization and/or wash at 1, 2, 3, or 4° C. lower than the thermalmelting point (T_(m)); moderately stringent conditions can utilize ahybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than thethermal melting point (T_(m)); low stringency conditions can utilize ahybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower thanthe thermal melting point (T_(m)). Using the equation, hybridization andwash compositions, and desired T_(m), those of ordinary skill willunderstand that variations in the stringency of hybridization and/orwash solutions are inherently described. If the desired degree ofmismatching results in a T_(m) of less than 45° C. (aqueous solution) or32° C. (formamide solution) it is preferred to increase the SSCconcentration so that a higher temperature can be used. An extensiveguide to the hybridization of nucleic acids is found in Tijssen,Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2“Overview of principles of hybridization and the strategy of nucleicacid probe assays”, Elsevier, New York (1993); and Current Protocols inMolecular Biology, Chapter 2, Ausubel et al., Eds., Greene Publishingand Wiley-Interscience, New York (1995). Hybridization and/or washconditions can be applied for at least 10, 30, 60, 90, 120, or 240minutes.

“Sequence identity” or “identity” in the context of nucleic acid orpolypeptide sequences refers to the nucleic acid bases or amino acidresidues in two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window.

Thus, “percentage of sequence identity” refers to the value determinedby comparing two optimally aligned sequences over a comparison window,wherein the portion of the polynucleotide or polypeptide sequence in thecomparison window may comprise additions or deletions (i.e., gaps) ascompared to the reference sequence (which does not comprise additions ordeletions) for optimal alignment of the two sequences. The percentage iscalculated by determining the number of positions at which the identicalnucleic acid base or amino acid residue occurs in both sequences toyield the number of matched positions, dividing the number of matchedpositions by the total number of positions in the window of comparisonand multiplying the results by 100 to yield the percentage of sequenceidentity. Useful examples of percent sequence identities include, butare not limited to, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%,or any integer percentage from 50% to 100%. These identities can bedetermined using any of the programs described herein.

Sequence alignments and percent identity or similarity calculations maybe determined using a variety of comparison methods designed to detecthomologous sequences including, but not limited to, the MegAlign™program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.,Madison, Wis.). Within the context of this application it will beunderstood that where sequence analysis software is used for analysis,that the results of the analysis will be based on the “default values”of the program referenced, unless otherwise specified. As used herein“default values” will mean any set of values or parameters thatoriginally load with the software when first initialized.

The “Clustal V method of alignment” corresponds to the alignment methodlabeled Clustal V (described by Higgins and Sharp, CABIOS. 5:151-153(1989); Higgins, D. G. et al., Comput. Appl. Biosci. 8:189-191 (1992))and found in the MegAlign™ program of the LASERGENE bioinformaticscomputing suite (DNASTAR Inc., Madison, Wis.). For multiple alignments,the default values correspond to GAP PENALTY=10 and GAP LENGTHPENALTY=10. Default parameters for pairwise alignments and calculationof percent identity of protein sequences using the Clustal V method areKTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleicacids these parameters are KTUPLE=2, GAP PENALTY=5, WINDOW=4 andDIAGONALS SAVED=4. After alignment of the sequences using the Clustal Vprogram, it is possible to obtain a “percent identity” by viewing the“sequence distances” table in the same program.

The “Clustal W method of alignment” corresponds to the alignment methodlabeled Clustal W (described by Higgins and Sharp, supra; Higgins, D. G.et al., supra) and found in the MegAlign™ v6.1 program of the LASERGENEbioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Defaultparameters for multiple alignment correspond to GAP PENALTY=10, GAPLENGTH PENALTY=0.2, Delay Divergen Seqs(%)=30, DNA TransitionWeight=0.5, Protein Weight Matrix=Gonnet Series, DNA Weight Matrix=IUB.After alignment of the sequences using the Clustal W program, it ispossible to obtain a “percent identity” by viewing the “sequencedistances” table in the same program.

“BLASTN method of alignment” is an algorithm provided by the NationalCenter for Biotechnology Information (NCBI) to compare nucleotidesequences using default parameters.

It is well understood by one skilled in the art that many levels ofsequence identity are useful in identifying polypeptides, from otherspecies, wherein such polypeptides have the same or similar function oractivity. Useful examples of percent identities include, but are notlimited to, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or anyinteger percentage from 50% to 100%. Indeed, any integer amino acididentity from 50% to 100% may be useful in describing the presentinvention, such as 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. Also, ofinterest is any full-length or partial complement of this isolatednucleotide fragment.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein and can include either the coding region alone or the codingregion in addition to the regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.“Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers to any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign” gene refers to a genenot normally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

The term “genome” as it applies to plant cells encompasses not onlychromosomal DNA found within the nucleus, but organelle DNA found withinsubcellular components (e.g., mitochondrial, plastid) of the cell.

A “codon-optimized gene” is a gene having its frequency of codon usagedesigned to mimic the frequency of preferred codon usage of the hostcell.

An “allele” is one of several alternative forms of a gene occupying agiven locus on a chromosome. When all the alleles present at a givenlocus on a chromosome are the same that plant is homozygous at thatlocus. If the alleles present at a given locus on a chromosome differthat plant is heterozygous at that locus.

“Coding sequence” refers to a DNA sequence that codes for a specificamino acid sequence. “Regulatory sequences” refer to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may include, butare not limited to: promoters, translation leader sequences, introns,polyadenylation recognition sequences, RNA processing sites, effectorbinding sites and stem-loop structures.

“Promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. The promoter sequenceconsists of proximal and more distal upstream elements, the latterelements often referred to as enhancers. Accordingly, an “enhancer” is aDNA sequence that can stimulate promoter activity, and may be an innateelement of the promoter or a heterologous element inserted to enhancethe level or tissue-specificity of a promoter. Promoters may be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic DNA segments. It is understood by those skilled inthe art that different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental conditions. It is furtherrecognized that since in most cases the exact boundaries of regulatorysequences have not been completely defined, DNA fragments of somevariation may have identical promoter activity. Promoters that cause agene to be expressed in most cell types at most times are commonlyreferred to as “constitutive promoters”. New promoters of various typesuseful in plant cells are constantly being discovered; numerous examplesmay be found in the compilation by Okamuro, J. K., and Goldberg, R. B.Biochemistry of Plants 15:1-82 (1989).

“Translation leader sequence” refers to a polynucleotide sequencelocated between the promoter sequence of a gene and the coding sequence.The translation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency. Examples of translation leadersequences have been described (Turner, R. and Foster, G. D., Mol.Biotechnol. 3:225-236 (1995)).

“3′ non-coding sequences”, “transcription terminator” or “terminationsequences” refer to DNA sequences located downstream of a codingsequence, including polyadenylation recognition sequences and othersequences encoding regulatory signals capable of affecting mRNAprocessing or gene expression. The polyadenylation signal is usuallycharacterized by affecting the addition of polyadenylic acid tracts tothe 3′ end of the mRNA precursor. The use of different 3′ non-codingsequences is exemplified by Ingelbrecht, I. L., et al. Plant Cell1:671-680 (1989).

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript. An RNA transcript is referred toas the mature RNA when it is an RNA sequence derived frompost-transcriptional processing of the primary transcript. “MessengerRNA” or “mRNA” refers to the RNA that is without introns and that can betranslated into protein by the cell. “cDNA” refers to a DNA that iscomplementary to, and synthesized from, an mRNA template using theenzyme reverse transcriptase. The cDNA can be single-stranded orconverted into double-stranded form using the Klenow fragment of DNApolymerase I. “Sense” RNA refers to RNA transcript that includes themRNA and can be translated into protein within a cell or in vitro.“Antisense RNA” refers to an RNA transcript that is complementary to allor part of a target primary transcript or mRNA, and that blocks orreduces the expression of a target gene (U.S. Pat. No. 5,107,065). Thecomplementarity of an antisense RNA may be with any part of the specificgene transcript, i.e., at the 5′ non-coding sequence, 3′ non-codingsequence, introns, or the coding sequence. “Functional RNA” refers toantisense RNA, ribozyme RNA, or other RNA that may not be translated butyet has an effect on cellular processes. The terms “complement” and“reverse complement” are used interchangeably herein with respect tomRNA transcripts, and are meant to define the antisense RNA of themessage.

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis affected by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of affecting the expression ofthat coding sequence (i.e., the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in a sense or antisenseorientation. In another example, the complementary RNA regions of theinvention can be operably linked, either directly or indirectly, 5′ tothe target mRNA, or 3′ to the target mRNA, or within the target mRNA, ora first complementary region is 5′ and its complement is 3′ to thetarget mRNA.

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook, J.,Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual;Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989).Transformation methods are well known to those skilled in the art andare described infra.

“PCR” or “polymerase chain reaction” is a technique for the synthesis oflarge quantities of specific DNA segments and consists of a series ofrepetitive cycles (Perkin Elmer Cetus Instruments, Norwalk, Conn.).Typically, the double-stranded DNA is heat denatured, the two primerscomplementary to the 3′ boundaries of the target segment are annealed atlow temperature and then extended at an intermediate temperature. Oneset of these three consecutive steps is referred to as a “cycle”.

The term “recombinant” refers to an artificial combination of twootherwise separated segments of sequence, e.g., by chemical synthesis orby the manipulation of isolated segments of nucleic acids by geneticengineering techniques.

A “plasmid” or “vector” is an extra chromosomal element often carryinggenes that are not part of the central metabolism of the cell, andusually in the form of circular double-stranded DNA fragments. Suchelements may be autonomously replicating sequences, genome integratingsequences, phage or nucleotide sequences, linear or circular, of asingle- or double-stranded DNA or RNA, derived from any source, in whicha number of nucleotide sequences have been joined or recombined into aunique construction which is capable of introducing an expressioncassette(s) into a cell. “Expression cassette” refers to a fragment ofDNA containing a foreign gene and having elements in addition to theforeign gene that allow for enhanced expression of that gene in aforeign host. “Transformation cassette” refers to a fragment of DNAcontaining a foreign gene and having elements in addition to the foreigngene that facilitate transformation of a particular host cell.

The terms “recombinant construct”, “expression construct”, “chimericconstruct”, “construct”, and “recombinant DNA construct” are usedinterchangeably herein. A recombinant construct comprises an artificialcombination of nucleic acid fragments, e.g., regulatory and codingsequences that are not found together in nature. For example, arecombinant construct may comprise regulatory sequences and codingsequences that are derived from different sources, or regulatorysequences and coding sequences derived from the same source, butarranged in a manner different than that found in nature. Such aconstruct may be used by itself or may be used in conjunction with avector. If a vector is used, then the choice of vector is dependent uponthe method that will be used to transform host cells as is well known tothose skilled in the art. For example, a plasmid vector can be used. Theskilled artisan is well aware of the genetic elements that must bepresent on the vector in order to successfully transform, select andpropagate host cells comprising any of the isolated nucleic acidfragments of the invention. The skilled artisan will also recognize thatdifferent independent transformation events will result in differentlevels and patterns of expression (Jones et al., EMBO J. 4:2411-2418(1985); De Almeida et al., Mol. Gen. Genetics 218:78-86 (1989)), andthus that multiple events must be screened in order to obtain linesdisplaying the desired expression level and pattern. Such screening maybe accomplished by Southern analysis of DNA, Northern analysis of mRNAexpression, immunoblotting analysis of protein expression, or phenotypicanalysis, among others.

The term “expression”, as used herein, refers to the production of afunctional end-product (e.g., an mRNA or a protein [either precursor ormature]).

The term “introduced” means providing a nucleic acid (e.g., expressionconstruct) or protein into a cell. Introduced includes reference to theincorporation of a nucleic acid into a eukaryotic or prokaryotic cellwhere the nucleic acid may be incorporated into the genome of the cell,and includes reference to the transient provision of a nucleic acid orprotein to the cell. Introduced includes reference to stable ortransient transformation methods, as well as sexually crossing. Thus,“introduced” in the context of inserting a nucleic acid fragment (e.g.,a recombinant construct/expression construct) into a cell, means“transfection” or “transformation” or “transduction” and includesreference to the incorporation of a nucleic acid fragment into aeukaryotic or prokaryotic cell where the nucleic acid fragment may beincorporated into the genome of the cell (e.g., chromosome, plasmid,plastid or mitochondrial DNA), converted into an autonomous replicon, ortransiently expressed (e.g., transfected mRNA).

“Mature” protein refers to a post-translationally processed polypeptide(i.e., one from which any pre- or propeptides present in the primarytranslation product have been removed). “Precursor” protein refers tothe primary product of translation of mRNA (i.e., with pre- andpropeptides still present). Pre- and propeptides may be but are notlimited to intracellular localization signals.

“Stable transformation” refers to the transfer of a nucleic acidfragment into a genome of a host organism, including both nuclear andorganellar genomes, resulting in genetically stable inheritance. Incontrast, “transient transformation” refers to the transfer of a nucleicacid fragment into the nucleus, or DNA-containing organelle, of a hostorganism resulting in gene expression without integration or stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” organisms.

As used herein, “transgenic” refers to a plant or a cell which compriseswithin its genome a heterologous polynucleotide. Preferably, theheterologous polynucleotide is stably integrated within the genome suchthat the polynucleotide is passed on to successive generations. Theheterologous polynucleotide may be integrated into the genome alone oras part of an expression construct. Transgenic is used herein to includeany cell, cell line, callus, tissue, plant part or plant, the genotypeof which has been altered by the presence of heterologous nucleic acidincluding those transgenics initially so altered as well as thosecreated by sexual crosses or asexual propagation from the initialtransgenic. The term “transgenic” as used herein does not encompass thealteration of the genome (chromosomal or extra-chromosomal) byconventional plant breeding methods or by naturally occurring eventssuch as random cross-fertilization, non-recombinant viral infection,non-recombinant bacterial transformation, non-recombinant transposition,or spontaneous mutation.

“Antisense inhibition” refers to the production of antisense RNAtranscripts capable of suppressing the expression of the target protein.“Co-suppression” refers to the production of sense RNA transcriptscapable of suppressing the expression of identical or substantiallysimilar foreign or endogenous genes (U.S. Pat. No. 5,231,020).Co-suppression constructs in plants previously have been designed byfocusing on overexpression of a nucleic acid sequence having homology toan endogenous mRNA, in the sense orientation, which results in thereduction of all RNA having homology to the overexpressed sequence(Vaucheret et al., Plant J. 16:651-659 (1998); Gura, Nature 404:804-808(2000)). The overall efficiency of this phenomenon is low, and theextent of the RNA reduction is widely variable. More recent work hasdescribed the use of “hairpin” structures that incorporate all, or part,of an mRNA encoding sequence in a complementary orientation that resultsin a potential “stem-loop” structure for the expressed RNA (PCTPublication No. WO 99/53050; PCT Publication No. WO 02/00904). Thisincreases the frequency of co-suppression in the recovered transgenicplants. Another variation describes the use of plant viral sequences todirect the suppression, or “silencing”, of proximal mRNA encodingsequences (PCT Publication No. WO 98/36083). Both of theseco-suppressing phenomena have not been elucidated mechanistically,although genetic evidence has begun to unravel this complex situation(Elmayan et al., Plant Cell 10:1747-1757 (1998)).

The term “oleaginous” refers to those organisms that tend to store theirenergy source in the form of lipid (Weete, In: Fungal LipidBiochemistry, 2nd Ed., Plenum, 1980). A class of plants identified asoleaginous are commonly referred to as “oilseed” plants. Examples ofoilseed plants include, but are not limited to: soybean (Glycine andSoja sp.), flax (Linum sp.), rapeseed (Brassica sp.), maize, cotton,safflower (Carthamus sp.) and sunflower (Helianthus sp.).

Within oleaginous microorganisms the cellular oil or TAG contentgenerally follows a sigmoid curve, wherein the concentration of lipidincreases until it reaches a maximum at the late logarithmic or earlystationary growth phase and then gradually decreases during the latestationary and death phases (Yongmanitchai and Ward, Appl. Environ.Microbiol. 57:419-25 (1991)). The term “oleaginous yeast” refers tothose microorganisms classified as yeasts that make oil. It is notuncommon for oleaginous microorganisms to accumulate in excess of about25% of their dry cell weight as oil. Examples of oleaginous yeastinclude, but are no means limited to, the following genera: Yarrowia,Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon andLipomyces.

As used herein, the term “biomass” refers specifically to spent or usedyeast cellular material resulting from the fermentation of a recombinantproduction host producing PUFAs in commercially significant amounts,wherein the preferred production host is a recombinant strain of theoleaginous yeast, Yarrowia lipolytica. The biomass may be in the form ofwhole cells, whole cell lysates, homogenized cells, partially hydrolyzedcellular material, and/or partially purified cellular material (e.g.,microbially produced oil).

The term “Euglenophyceae” refers to a group of unicellular colorless orphotosynthetic flagellates (“euglenoids”) found living in freshwater,marine, soil, and parasitic environments. The class is characterized bysolitary unicells, wherein most are free-swimming and have two flagella(one of which may be nonemergent) arising from an anterior invaginationknown as a reservoir. Photosynthetic euglenoids contain one to manygrass-green chloroplasts, which vary from minute disks to expandedplates or ribbons. Colorless euglenoids depend on osmotrophy orphagotrophy for nutrient assimilation. About 1000 species have beendescribed and classified into about 40 genera and 6 orders. Examples ofEuglenophyceae include, but are by no means limited to, the followinggenera: Euglena, Eutreptiella and Tetruetreptia.

The term “plant” refers to whole plants, plant organs, plant tissues,seeds, plant cells, seeds and progeny of the same. Plant cells include,without limitation, cells from seeds, suspension cultures, embryos,meristematic regions, callus tissue, leaves, roots, shoots,gametophytes, sporophytes, pollen and microspores.

“Progeny” comprises any subsequent generation of a plant.

An Overview: Microbial Biosynthesis of Fatty Acids and Triacylglycerols

In general, lipid accumulation in oleaginous microorganisms is triggeredin response to the overall carbon to nitrogen ratio present in thegrowth medium. This process, leading to the de novo synthesis of freepalmitate (16:0) in oleaginous microorganisms, is described in detail inU.S. Pat. No. 7,238,482. Palmitate is the precursor of longer-chainsaturated and unsaturated fatty acid derivates, which are formed throughthe action of elongases and desaturases (FIG. 1).

TAGs (the primary storage unit for fatty acids) are formed by a seriesof reactions that involve: (1) the esterification of one molecule ofacyl-CoA to glycerol-3-phosphate via an acyltransferase to producelysophosphatidic acid; (2) the esterification of a second molecule ofacyl-CoA via an acyltransferase to yield 1,2-diacylglycerol phosphate(commonly identified as phosphatidic acid); (3) removal of a phosphateby phosphatidic acid phosphatase to yield 1,2-diacylglycerol (DAG); and(4) the addition of a third fatty acid by the action of anacyltransferase to form TAG. A wide spectrum of fatty acids can beincorporated into TAGs, including saturated and unsaturated fatty acidsand short-chain and long-chain fatty acids.

Biosynthesis of Omega Fatty Acids

The metabolic process wherein oleic acid is converted to long chainomega-3/omega-6 fatty acids involves elongation of the carbon chainthrough the addition of carbon atoms and desaturation of the moleculethrough the addition of double bonds. This requires a series of specialdesaturation and elongation enzymes present in the endoplasmic reticulummembrane. However, as seen in FIG. 1 and as described below, there areoften multiple alternate pathways for production of a specific longchain omega-3/omega-6 fatty acid.

Specifically, all pathways require the initial conversion of oleic acidto LA, the first of the omega-6 fatty acids, by a delta-12 desaturase.Then, using the “delta-9 elongase/delta-8 desaturase pathway” and LA assubstrate, long chain omega-6 fatty acids are formed as follows: (1) LAis converted to EDA by a delta-9 elongase; (2) EDA is converted to DGLAby a delta-8 desaturase; (3) DGLA is converted to ARA by a delta-5desaturase; (4) ARA is converted to DTA by a C_(20/22) elongase; and,(5) DTA is converted to DPAn-6 by a delta-4 desaturase. Alternatively,the “delta-9 elongase/delta-8 desaturase pathway” can use ALA assubstrate to produce long chain omega-3 fatty acids as follows: (1) LAis converted to ALA, the first of the omega-3 fatty acids, by a delta-15desaturase; (2) ALA is converted to ETrA by a delta-9 elongase; (3) ETrAis converted to ETA by a delta-8 desaturase; (4) ETA is converted to EPAby a delta-5 desaturase; (5) EPA is converted to DPA by a C_(20/22)elongase; and (6) DPA is converted to DHA by a delta-4 desaturase.Optionally, omega-6 fatty acids may be converted to omega-3 fatty acids;for example, ETA and EPA are produced from DGLA and ARA, respectively,by delta-17 desaturase activity.

Alternate pathways for the biosynthesis of omega-3/omega-6 fatty acidsutilize a delta-6 desaturase and C_(18/20) elongase (also known asdelta-6 elongase, the terms can be used interchangeably) (i.e., the“delta-6 desaturase/delta-6 elongase pathway”). More specifically, LAand ALA may be converted to GLA and STA, respectively, by a delta-6desaturase; then, a C_(18/20) elongase converts GLA to DGLA and/or STAto ETA.

It is contemplated that the particular functionalities required to beintroduced into a specific host organism for production ofomega-3/omega-6 fatty acids will depend on the host cell (and its nativePUFA profile and/or desaturase/elongase profile), the availability ofsubstrate, and the desired end product(s). For example, expression ofthe delta-9 elongase/delta-8 desaturase pathway may be preferred in someembodiments, as opposed to expression of the delta-6 desaturase/delta-6elongase pathway, since PUFAs produced via the former pathway are devoidof GLA.

One skilled in the art will be able to identify various candidate genesencoding each of the enzymes desired for omega-3/omega-6 fatty acidbiosynthesis. Useful desaturase and elongase sequences may be derivedfrom any source, e.g., isolated from a natural source (from bacteria,algae, fungi, plants, animals, etc.), produced via a semi-syntheticroute or synthesized de novo. Although the particular source of thedesaturase and elongase genes introduced into the host is not critical,considerations for choosing a specific polypeptide having desaturase orelongase activity include: (1) the substrate specificity of thepolypeptide; (2) whether the polypeptide or a component thereof is arate-limiting enzyme; (3) whether the desaturase or elongase isessential for synthesis of a desired PUFA; (4) co-factors required bythe polypeptide; and/or, (5) whether the polypeptide is modified afterits production (e.g., by a kinase or a prenyltransferase). The expressedpolypeptide preferably has parameters compatible with the biochemicalenvironment of its location in the host cell (see U.S. Pat. No.7,238,482 for additional details).

In additional embodiments, it will also be useful to consider theconversion efficiency of each particular desaturase and/or elongase.More specifically, since each enzyme rarely functions with 100%efficiency to convert substrate to product, the final lipid profile ofunpurified oils produced in a host cell will typically be a mixture ofvarious PUFAs consisting of the desired omega-3/omega-6 fatty acid, aswell as various upstream intermediary PUFAs. Thus, each enzyme'sconversion efficiency is also a variable to consider when optimizingbiosynthesis of a desired fatty acid.

With each of the considerations above in mind, candidate genes havingthe appropriate desaturase and elongase activities (e.g., delta-6desaturases, C_(18/20) elongases, delta-5 desaturases, delta-17desaturases, delta-15 desaturases, delta-9 desaturases, delta-12desaturases, C_(14/16) elongases, C_(16/18) elongases, delta-9elongases, delta-8 desaturases, delta-4 desaturases, C_(20/22) elongasesand DHA synthases) can be identified according to publicly availableliterature (e.g., GenBank), the patent literature, and experimentalanalysis of organisms having the ability to produce PUFAs. These geneswill be suitable for introduction into a specific host organism, toenable or enhance the organism's synthesis of PUFAs.

Multizymes and Linkers

In one embodiment, the present invention concerns a multizyme comprisinga single polypeptide having at least two independent and separableenzymatic activities

Examples of suitable enzymatic activities include elongases, fatty aciddesaturases, transferases, acyl CoA synthases and thioesterases. Forexample, suitable fatty acid desaturases include, but are not limitedto: delta-4 desaturase, delta-5 desaturase, delta-6 desaturase, delta-8desaturase, delta-9 desaturase, delta-12 desaturase, delta-15desaturase, and/or delta-17 desaturase. Examples of suitable elongasesinclude, but are not limited to: delta-9 elongase, C_(14/16) elongase,C_(16/18) elongase, C_(18/20) elongase, and/or C_(20/22) elongase.

Examples of suitable transferases include but are not limited to acyltransferases such as glycerol-3-phosphate O-acyltransferase (also calledglycerol-phosphate acyl transferase or glycerol-3-phosphate acyltransferase; GPAT), 2-acylglycerol O-acyltransferase,1-acylglycerol-3-phosphate O-acyltransferase (also called1-acylglycerol-phosphate acyltransferase or lyso-phosphatidic acidacyltransferase; AGPAT or LPAAT or LPAT), 2-acylglycerol-3-phosphateO-acyltransferase, 1-acylglycerophosphocholine O-acyltransferase (alsocalled lyso-lecithin acyltransferase or lyso-phosphatidylcholineacyltransferase; AGPCAT or LLAT or LPCAT), 2-acylglycerophosphocholineO-acyltransferase, diacylglycerol O-acyltransferase (also calleddiglyceride acyltransferase; DAGAT or DGAT) andphospholipid:diacylglycerol acyltransferase (PDAT).

An example of a suitable acyl CoA synthetase includes but is not limitedto long-chain-fatty-acid-CoA ligase (also called acyl-activating enzymeor acyl-CoA synthetase).

An example of a suitable thioesterase includes but is not limited tooleoyl-[acyl-carrier-protein] hydrolase (also calledacyl-[acyl-carrier-protein] hydrolase, acyl-ACP-hydrolase oracyl-ACP-thioesterase).

Preferably, the instant multizyme should have enzymatic activitiescomprising at least one fatty acid elongase linked to at least one fattyacid desaturase.

The link used to form the multizyme is minimally comprised of a singlepolypeptide bond. In another aspect, the link may be comprised of oneamino acid residue, such as proline, or a polypeptide. It may bedesirable that if the link is a polypeptide then it has at least oneproline amino acid residue.

Preferably, the multizyme of the invention comprises a first enzymaticactivity linked to a second enzymatic activity and the link is selectedfrom the group consisting of a polypeptide bond, SEQ ID NO:198(EgDHAsyn1 linker), SEQ ID NO:200 (EgDHAsyn2 linker), SEQ ID NO:235(EaDHAsyn1 linker), SEQ ID NO:472, SEQ ID NO:504, and modified Yarrowialipolytica linkers (SEQ ID NOs:438 and 445).

Also within the scope of this invention is a method for making amultizyme which comprises:

-   -   (a) linking a first polypeptide with at least a second        polypeptide wherein each polypeptide has an independent and        separable enzymatic activity; and    -   (b) evaluating the product of step (a) for the independent and        separable enzymatic activities.

As was discussed above, the enzymatic activities are selected from thegroup consisting of fatty acid elongases, fatty acid desaturases, acyltransferases, acyl CoA synthases and thioesterases. Preferably, theenzymatic activities comprise at least one fatty acid elongase linked toat least one fatty acid desaturase.

Examples of suitable desaturases, elongases and linkers are discussedabove.

Although numerous examples of multizymes are described above, DHAsynthases (comprising both C20 elongase activity and delta-4 desaturaseactivity) and DGLA synthases (comprising both delta-9 elongase anddelta-8 desaturase activity) are of particular interest. Data describedherein confirm that linking of the two domains within each synthaseresults in increased efficiency or flux, as compared to efficiency orflux observed when the enzymatic domains exist as independent entities,i.e., not linked together in a multizyme.

For example, when a mulltizyme comprising the Euglena gracilis C20elongase domain and a Schizochytrium aggregatum delta-4 desaturase wasexpressed in Yarrowia lipolytica, the delta-4 desaturase activity wasapproximately 2 to 3-fold greater in the fused construct, as opposed toits activity when expressed alone (Example 28). Similarly, when theEuglena gracilis C20 elongase domain-Schizochytrium aggregatum delta-4desaturase fusion was expressed as a multizyme in soybean, increased EPAto DHA flux was measured, as opposed to when the two enzymes wereexpressed independently (Example 49).

Increased efficiency (or LA to DGLA flux) was also demonstrated invarious DGLA synthases that were created. A series of six delta-9elongase/delta-8 desaturase fusion constructs were created using variouscombinations of delta-9 elongases derived from E. gracillis, E. anabaenaUTEX 373 and Eutreptiella sp. CCMP389 and delta-8 desaturases derivedfrom E. gracillis and E. anabaena UTEX 373; these were individuallyexpressed in Yarrowia lipolytica (Examples 55 and 56, respectively). Inall cases, the fusion gene had higher activity than the individual genealone when expressed in Yarrowia. These data again suggested that theproduct of delta-9 elongase may be directly channeled as substrate ofdelta-8 desaturase in the fusion protein. One skilled in the art wouldbe able to use the teachings herein to create various other multizymesthat have increased efficiency or flux. Accordingly, the inventionrelates to any multizyme that is made using a linker derived from thesequences of the invention. Preferred multizymes are those that combinevarious genes of the PUFA biosynthetic pathway.

Sequence Identification of Novel DHA Synthases

In the present invention, nucleotide sequences encoding DHA synthaseshave been isolated from Euglena gracilis and Euglena anabaena, assummarized below in Table 4.

TABLE 4 Summary Of Euglena DHA Synthases DHA Synthase Nucleotide AminoAcid Designation Organism SEQ ID NO SEQ ID NO EgDHAsyn1 E. gracilis 1112 EgDHAsyn1* E. gracilis 205 12 EgDHAsyn2 E. gracilis 21 22 EaDHAsyn1E. anabaena 91 95 EaDHAsyn2 E. anabaena 92 96 EaDHAsyn3 E. anabaena 9397 EgDHAsyn1S Synthetically 410 411 (codon-optimized for derived from(identical to Yarrowia expression) E. gracilis SEQ ID EgDHAsyn1 NO: 12)

In some embodiments, the instant EgDHAsyn1, EgDHAsyn2, EaDHAsyn1,EaDHAsyn2 and EaDHAsyn3 DHA synthase sequences can be codon-optimizedfor expression in a particular host organism. As is well known in theart, this can be a useful means to further optimize the expression ofthe enzyme in the alternate host, since use of host-preferred codons cansubstantially enhance the expression of the foreign gene encoding thepolypeptide. EgDHAsyn1, for example, was codon-optimized for expressionin Yarrowia lipolytica (example 54), thereby yielding EgDHAsyn1S (astaught in U.S. Pat. No. 7,238,482 and U.S. Pat. No. 7,125,672).

One skilled in the art would be able to use the teachings herein tocreate various other codon-optimized DHA synthase proteins suitable foroptimal expression in alternate hosts, based on the wildtype EgDHAsyn1,EgDHAsyn2, EaDHAsyn1, EaDHAsyn2 and/or EaDHAsyn3 sequences describedabove in Table 4. Accordingly, the instant invention relates to anycodon-optimized DHA synthase protein that is derived from a wildtypesequence of the instant invention. In some preferred embodiments, it maybe desirable to modify a portion of the codons encoding EgDHAsyn1,EgDHAsyn2, EaDHAsyn1, EaDHAsyn2 and/or EaDHAsyn3 to enhance expressionof the gene in a host organism including, but not limited to, a plant orplant part.

In another embodiment, the present invention concerns an isolatedpolynucleotide encoding a DHA synthase comprising:

-   -   (a) a nucleotide sequence encoding a polypeptide having DHA        synthase activity, wherein the polypeptide has at least 80%        amino acid identity, based on the Clustal V method of alignment,        when compared to an amino acid sequence as set forth in SEQ ID        NO:12, SEQ ID NO:22, SEQ ID NO:95, SEQ ID NO:96, or SEQ ID        NO:97;    -   (b) a nucleotide sequence encoding a polypeptide having DHA        synthase activity wherein the nucleotide sequence has at least        80% sequence identity, based on the BLASTN method of alignment,        when compared to a nucleotide sequence as set forth in SEQ ID        NO:11, SEQ ID NO:205, SEQ ID NO:21, SEQ ID NO:91, SEQ ID NO:92,        SEQ ID NO:93, or SEQ ID NO:410;    -   (c) a nucleotide sequence encoding a polypeptide having DHA        synthase activity, wherein the nucleotide sequence hybridizes        under stringent conditions to a nucleotide sequence as set forth        in SEQ ID NO:11, SEQ ID NO:21, SEQ ID NO:91, SEQ ID NO:92, SEQ        ID NO:93, SEQ ID NO:205, or SEQ ID NO:410; or    -   (d) a complement of the nucleotide sequence of (a), (b) or (c),        wherein the complement and the nucleotide sequence consist of        the same number of nucleotides and are 100% complementary.

In still another aspect, this invention concerns an isolatedpolynucleotide comprising:

-   -   (a) a nucleotide sequence encoding a polypeptide having DHA        synthase activity, wherein the polypeptide has at least 80%        amino acid identity, based on the Clustal V method of alignment,        when compared to an amino acid sequence as set forth in SEQ ID        NO:12, SEQ ID NO:22, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97,        or SEQ ID NO:411;    -   (b) a nucleotide sequence encoding a polypeptide having DHA        synthase activity wherein the nucleotide sequence has at least        80% sequence identity, based on the BLASTN method of alignment,        when compared to a nucleotide sequence as set forth in SEQ ID        NO:11, SEQ ID NO:205, SEQ ID NO:21, SEQ ID NO:91, SEQ ID NO:92,        SEQ ID NO:93 or SEQ ID NO:410;    -   (c) a nucleotide sequence encoding a polypeptide having DHA        synthase activity, wherein the nucleotide sequence hybridizes        under stringent conditions to a nucleotide sequence as set forth        in SEQ ID NO:11, SEQ ID NO:205, SEQ ID NO:21, SEQ ID NO:91, SEQ        ID NO:92, SEQ ID NO:93 or SEQ ID NO:410; or    -   (d) a complement of the nucleotide sequence of (a), (b) or (c),        wherein the complement and the nucleotide sequence consist of        the same number of nucleotides and are 100% complementary.

Preferably, an isolated polynucleotide encoding a DHA synthase comprisesthe sequence set forth in any of SEQ ID NO:11, SEQ ID NO:205, SEQ IDNO:21, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, or SEQ ID NO:410.

Identification and Isolation of Homologs

Any of the instant DHA synthase sequences (i.e., EgDHAsyn1, EgDHAsyn2,EaDHAsyn1, EaDHAsyn2 and EaDHAsyn3) or portions thereof may be used tosearch for DHA synthase homologs in the same or other bacterial, algal,fungal, euglenoid or plant species using sequence analysis software. Ingeneral, such computer software matches similar sequences by assigningdegrees of homology to various substitutions, deletions, and othermodifications.

Alternatively, any of the instant DHA synthase sequences or portionsthereof may also be employed as hybridization reagents for theidentification of DHA synthase homologs. The basic components of anucleic acid hybridization test include a probe, a sample suspected ofcontaining the gene or gene fragment of interest and a specifichybridization method. Probes of the present invention are typicallysingle-stranded nucleic acid sequences that are complementary to thenucleic acid sequences to be detected. Probes are “hybridizable” to thenucleic acid sequence to be detected. Although the probe length can varyfrom 5 bases to tens of thousands of bases, typically a probe length ofabout 15 bases to about 30 bases is suitable. Only part of the probemolecule needs to be complementary to the nucleic acid sequence to bedetected. In addition, the complementarity between the probe and thetarget sequence need not be perfect. Hybridization does occur betweenimperfectly complementary molecules with the result that a certainfraction of the bases in the hybridized region are not paired with theproper complementary base.

Hybridization methods are well defined. Typically the probe and samplemust be mixed under conditions that will permit nucleic acidhybridization. This involves contacting the probe and sample in thepresence of an inorganic or organic salt under the proper concentrationand temperature conditions. The probe and sample nucleic acids must bein contact for a long enough time that any possible hybridizationbetween the probe and sample nucleic acid may occur. The concentrationof probe or target in the mixture will determine the time necessary forhybridization to occur. The higher the probe or target concentration,the shorter the hybridization incubation time needed. Optionally, achaotropic agent may be added (e.g., guanidinium chloride, guanidiniumthiocyanate, sodium thiocyanate, lithium tetrachloroacetate, sodiumperchlorate, rubidium tetrachloroacetate, potassium iodide, cesiumtrifluoroacetate). If desired, one can add formamide to thehybridization mixture, typically 30-50% (v/v).

Various hybridization solutions can be employed. Typically, thesecomprise from about 20 to 60% volume, preferably 30%, of a polar organicsolvent. A common hybridization solution employs about 30-50% v/vformamide, about 0.15 to 1 M sodium chloride, about 0.05 to 0.1 Mbuffers (e.g., sodium citrate, Tris-HCl, PIPES or HEPES (pH range about6-9)), about 0.05 to 0.2% detergent (e.g., sodium dodecylsulfate), orbetween 0.5-20 mM EDTA, FICOLL (Pharmacia Inc.) (about 300-500 kdal),polyvinylpyrrolidone (about 250-500 kdal), and serum albumin. Alsoincluded in the typical hybridization solution will be unlabeled carriernucleic acids from about 0.1 to 5 mg/mL, fragmented nucleic DNA (e.g.,calf thymus or salmon sperm DNA, or yeast RNA), and optionally fromabout 0.5 to 2% wt/vol glycine. Other additives may also be included,such as volume exclusion agents that include a variety of polarwater-soluble or swellable agents (e.g., polyethylene glycol), anionicpolymers (e.g., polyacrylate or polymethylacrylate) and anionicsaccharidic polymers (e.g., dextran sulfate).

Nucleic acid hybridization is adaptable to a variety of assay formats.One of the most suitable is the sandwich assay format. The sandwichassay is particularly adaptable to hybridization under non-denaturingconditions. A primary component of a sandwich-type assay is a solidsupport. The solid support has adsorbed to it or covalently coupled toit an immobilized nucleic acid probe that is unlabeled and complementaryto one portion of the sequence.

In additional embodiments, any of the DHA synthase nucleic acidfragments described herein (or any homologs identified thereof) may beused to isolate genes encoding homologous proteins from the same orother bacterial, algal, fungal, euglenoid or plant species. Isolation ofhomologous genes using sequence-dependent protocols is well known in theart. Examples of sequence-dependent protocols include, but are notlimited to: (1) methods of nucleic acid hybridization; (2) methods ofDNA and RNA amplification, as exemplified by various uses of nucleicacid amplification technologies [e.g., polymerase chain reaction (PCR),Mullis et al., U.S. Pat. No. 4,683,202; ligase chain reaction (LCR),Tabor et al., Proc. Acad. Sci. USA 82:1074 (1985); or stranddisplacement amplification (SDA), Walker et al., Proc. Natl. Acad. Sci.U.S.A., 89:392 (1992)]; and (3) methods of library construction andscreening by complementation.

For example, genes encoding similar proteins or polypeptides to amultizyme or an individual domain thereof (such as the DHA synthases)described herein, could be isolated directly by using all or a portionof the instant nucleic acid fragments as DNA hybridization probes toscreen libraries from e.g., any desired yeast or fungus usingmethodology well known to those skilled in the art (wherein thoseorganisms producing DTA, DPAn-6, DPA and/or DHA would be preferred).Specific oligonucleotide probes based upon the instant nucleic acidsequences can be designed and synthesized by methods known in the art(Maniatis, supra). Moreover, the entire sequences can be used directlyto synthesize DNA probes by methods known to the skilled artisan (e.g.,random primers DNA labeling, nick translation or end-labelingtechniques), or RNA probes using available in vitro transcriptionsystems. In addition, specific primers can be designed and used toamplify a part of (or full-length of) the instant sequences. Theresulting amplification products can be labeled directly duringamplification reactions or labeled after amplification reactions, andused as probes to isolate full-length DNA fragments under conditions ofappropriate stringency.

Typically, in PCR-type amplification techniques, the primers havedifferent sequences and are not complementary to each other. Dependingon the desired test conditions, the sequences of the primers should bedesigned to provide for both efficient and faithful replication of thetarget nucleic acid. Methods of PCR primer design are common and wellknown in the art (Thein and Wallace, “The use of oligonucleotide asspecific hybridization probes in the Diagnosis of Genetic Disorders”, inHuman Genetic Diseases: A Practical Approach, K. E. Davis Ed., (1986) pp33-50, IRL: Herndon, Va.; and Rychlik, W., In Methods in MolecularBiology, White, B. A. Ed., (1993) Vol. 15, pp 31-39, PCR Protocols:Current Methods and Applications. Humania: Totowa, N.J.).

Generally two short segments of the instant sequences may be used in PCRprotocols to amplify longer nucleic acid fragments encoding homologousgenes from DNA or RNA. PCR may also be performed on a library of clonednucleic acid fragments wherein the sequence of one primer is derivedfrom the instant nucleic acid fragments, and the sequence of the otherprimer takes advantage of the presence of the polyadenylic acid tractsto the 3′ end of the mRNA precursor encoding eukaryotic genes.

Alternatively, the second primer sequence may be based upon sequencesderived from the cloning vector. For example, the skilled artisan canfollow the RACE protocol (Frohman et al., Proc. Natl. Acad. Sci. U.S.A.,85:8998 (1988)) to generate cDNAs by using PCR to amplify copies of theregion between a single point in the transcript and the 3′ or 5′ end.Primers oriented in the 3′ and 5′ directions can be designed from theinstant sequences. Using commercially available 3′ RACE or 5′ RACEsystems (Gibco/BRL, Gaithersburg, Md.), specific 3′ or 5′ cDNA fragmentscan be isolated (Ohara et al., Proc. Natl. Acad. Sci. U.S.A., 86:5673(1989); Loh et al., Science 243:217 (1989)).

In other embodiments, any of the enzymes (e.g., multizymes, DHAsynthases, or individual domains described herein) may be modified. Asis well known to those skilled in the art, in vitro mutagenesis andselection, chemical mutagenesis, “gene shuffling” methods or other meanscan be employed to obtain mutations of naturally occurring genes.Alternatively, multizymes may be synthesized by domain swapping, whereina functional domain from any enzyme may be exchanged with or added to afunctional domain in an alternate enzyme to thereby result in a novelprotein.

Sequence Identification of Novel C20 Elongases

In the present invention, nucleotide sequences encoding C20 elongaseshave been isolated from Euglena gracilis and Euglena anabaena, assummarized below in Table 5.

TABLE 5 Summary Of Euglena C20 Elongases C20 Elongase Nucleotide AminoAcid Designation Organism SEQ ID NO SEQ ID NO EgDHAsyn1 C20 E. gracilis201 202 elongase domain EgDHAsyn1* C20 E. gracilis 206 — elongase domainEgDHAsyn2 C20 E. gracilis 203 204 elongase domain EaDHAsyn1 C20 E.anabaena 227 231 elongase domain EaDHAsyn2 C20 E. anabaena 228 232elongase domain EaDHAsyn3 C20 E. anabaena 229 233 elongase domainEaDHAsyn4 C20 E. anabaena 230 — elongase domain EgC20ES C20Synthetically 183 184 elongase domain derived from (identical to(codon-optimized E. gracilis SEQ ID for expression in EgDHAsyn1 NO: 202)Yarrowia) EaC20ES C20 Synthetically 188 189 elongase domain derived from(identical to (codon-optimized E. anabaena SEQ ID for expression inEaDHAsyn2 NO: 232) Yarrowia)

The instant invention concerns an isolated polynucleotide encoding a C20elongase comprising:

-   -   (a) a nucleotide sequence encoding a polypeptide having C20        elongase activity, wherein the polypeptide has at least 80%        amino acid identity, based on the Clustal V method of alignment,        when compared to an amino acid sequence as set forth in SEQ ID        NO:12, SEQ ID NO:22, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97;        SEQ ID NO:202, SEQ ID NO:204, SEQ ID NO:231, SEQ ID NO:232, or        SEQ ID NO:233;    -   (b) a nucleotide sequence encoding a polypeptide having C20        elongase activity wherein the nucleotide sequence has at least        80% sequence identity, based on the BLASTN method of alignment,        when compared to a nucleotide sequence as set forth in SEQ ID        NO:11, SEQ ID NO:21, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93,        SEQ ID NO:183, SEQ ID NO:188, SEQ ID NO:201, SEQ ID NO:206, SEQ        ID NO:203, SEQ ID NO:227, SEQ ID NO:228, SEQ ID NO:229, or SEQ        ID NO:230;    -   (c) a nucleotide sequence encoding a polypeptide having C20        elongase activity, wherein the nucleotide sequence hybridizes        under stringent conditions to a nucleotide sequence as set forth        in SEQ ID NO:11, SEQ ID NO:21, SEQ ID NO:91, SEQ ID NO:92, SEQ        ID NO:93, SEQ ID NO:183, SEQ ID NO:188, SEQ ID NO:201, SEQ ID        NO:206, SEQ ID NO:203, SEQ ID NO:227, SEQ ID NO:228, SEQ ID        NO:229, SEQ ID NO:230; or    -   (d) a complement of the nucleotide sequence of (a), (b) or (c),        wherein the complement and the nucleotide sequence consist of        the same number of nucleotides and are 100% complementary.

Preferably, an isolated polynucleotide encoding a C20 elongase,comprises the sequence set forth in any of SEQ ID NO:183, SEQ ID NO:188,SEQ ID NO:201, SEQ ID NO:206, SEQ ID NO:203, SEQ ID NO:227, SEQ IDNO:228, SEQ ID NO:229, or SEQ ID NO:230.

Sequence Identification of Novel Delta-4 Desaturases

In the present invention, nucleotide sequences encoding delta-4desaturases have been isolated from Euglena gracilis and Euglenaanabaena, as summarized below in Table 6.

TABLE 6 Summary Of Euglena Delta-4 Desaturases Delta-4 DesaturaseNucleotide Amino Acid Designation* Organism SEQ ID NO SEQ ID NOEgDHAsyn1 delta-4 E. gracilis 214 215 desaturase domain 1 EgDHAsyn1delta-4 Synthetically 216 217 desaturase domain 1* derived from E.gracilis EgDHAsyn1 EgDHAsyn1* delta-4 E. gracilis 220 221 desaturasedomain 2 EaDHAsyn1 delta-4 E. anabaena 236 239 desaturase domain 1EaDHAsyn2 delta-4 E. anabaena 237 240 desaturase domain 1 EaDHAsyn4delta-4 E. anabaena 238 241 desaturase domain 1 EaDHAsyn1 delta-4 E.anabaena 242 246 desaturase domain 2 EaDHAsyn2 delta-4 E. anabaena 243247 desaturase domain 2 EaDHAsyn3 delta-4 E. anabaena 244 248 desaturasedomain 2 EaDHAsyn4 delta-4 E. anabaena 245 249 desaturase domain 2 EaD4Sdelta-4 Synthetically 192 193 desaturase domain derived from(codon-optimized for E. anabaena expression in EaDHAsyn2 Yarrowia) EgD4Sdelta-4 Synthetically 387 388 desaturase domain derived from(codon-optimized for E. gracilis expression in EgDHAsyn1 Yarrowia)*Note: The delta-4 desaturase domain 1 does not include the proline-richlinker of the DHA synthase from which it was derived. In contrast, thedelta-4 desaturase domain 2 does include the proline-rich linker of theDHA synthase from which it was derived.

In alternate embodiments, the instant delta-4 desaturase domainsequences can be codon-optimized for expression in a particular hostorganism. For example, the Euglena anabaena delta-4 desaturase domain ofEaDHAsyn2 was codon-optimized for expression in Yarrowia lipolytica. Forexample, the Euglena gracilis delta-4 desaturase domain of EgDHAsyn1 wasalso codon-optimized for expression in Yarrowia lipolytica One skilledin the art would be able to use the teachings herein to create variousother codon-optimized delta-4 desaturase proteins suitable for optimalexpression in alternate hosts, based on the wildtype delta-4 desaturasedomain sequences of EgDHAsyn1, EgDHAsyn2, EaDHAsyn1, EaDHAsyn2 and/orEaDHAsyn3 as described above in Table 6. Accordingly, the instantinvention relates to any codon-optimized delta-4 desaturase protein thatis derived from a wildtype sequence of the instant invention. In somepreferred embodiments, it may be desirable to modify a portion of thecodons encoding the delta-4 desaturase domain sequences of EgDHAsyn1,EgDHAsyn2, EaDHAsyn1, EaDHAsyn2 and/or EaDHAsyn3 to enhance expressionof the gene in a host organism including, but not limited to, a plant orplant part.

Moreover, based on the observation that the C-terminal portion of theC20 elongase domain of the DHA synthases appears to overlap with theN-terminal portion of the delta-4 desaturase domain, functional analyseswere performed to define the optimal functional delta-4 desaturasedomain. As described in Examples 51 and 53 hereinbelow, deletionmutagenesis studies were performed using the codon-optimized proteinsequences, EaD4S (SEQ ID NO:193) and EgD4S (SEQ ID NO:388). Thefollowing variants were produced: EaD4S-3 (SEQ ID NO:386), EaD4S-2 (SEQID NO:384), EaD4S-1 (SEQ ID NO:382), EgD4S-3 (SEQ ID NO:408), EgD4S-2(SEQ ID NO:406) and EgD4S-1 (SEQ ID NO:404).

One skilled in the art will recognize that since the exact boundaries ofthese particular delta-4 desaturase sequences from Euglena gracilis andEuglena anabaena have not been completely defined, protein fragments orpolypeptides of increased or diminished lengths may have comparabledelta-4 desaturase activity. Similarly, comparable truncations couldreadily be performed based on the wildtype delta-4 desaturase domainsequences of EgDHAsyn1, EgDHAsyn2, EaDHAsyn1, EaDHAsyn2 and/or EaDHAsyn3as described above in Table 6, to produce a delta-4 desaturase having asufficient amount of delta-4 desaturase activity, wherein equivalent orincreased delta-4 desaturase activity would be preferred.

Thus, the instant invention further concerns an isolated polynucleotideencoding a delta-4 desaturase comprising:

-   -   (a) a nucleotide sequence encoding a polypeptide having delta-4        desaturase activity, wherein the polypeptide has at least 80%        amino acid identity, based on the Clustal V method of alignment,        when compared to an amino acid sequence as set forth in SEQ ID        NO:12, SEQ ID NO:22, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97,        SEQ ID NO:215, SEQ ID NO:217, SEQ ID NO:221, SEQ ID NO:239, SEQ        ID NO:240, SEQ ID NO:241, SEQ ID NO:246, SEQ ID NO:247, SEQ ID        NO:248, SEQ ID NO:249, SEQ ID NO:193, SEQ ID NO:382, SEQ ID        NO:384, SEQ ID NO:386, SEQ ID NO:388, SEQ ID NO:404, SEQ ID        NO:406, or SEQ ID NO:408;    -   (b) a nucleotide sequence encoding a polypeptide having delta-4        desaturase activity wherein the nucleotide sequence has at least        80% sequence identity, based on the BLASTN method of alignment,        when compared to a nucleotide sequence as set forth in SEQ ID        NO:11, SEQ ID NO:21, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93,        SEQ ID NO:214, SEQ ID NO:216, SEQ ID NO:220, SEQ ID NO:236, SEQ        ID NO:237, SEQ ID NO:238, SEQ ID NO:242, SEQ ID NO:243, SEQ ID        NO:244, SEQ ID NO:245, SEQ ID NO:192, SEQ ID NO:381, SEQ ID        NO:383, SEQ ID NO:385, SEQ ID NO:387, SEQ ID NO:403, SEQ ID        NO:405 or SEQ ID NO:407;    -   (c) a nucleotide sequence encoding a polypeptide having delta-4        desaturase activity, wherein the nucleotide sequence hybridizes        under stringent conditions to a nucleotide sequence as set forth        in SEQ ID NO:11, SEQ ID NO:21, SEQ ID NO:91, SEQ ID NO:92, SEQ        ID NO:93, SEQ ID NO:214, SEQ ID NO:216, SEQ ID NO:220, SEQ ID        NO:236, SEQ ID NO:237, SEQ ID NO:238, SEQ ID NO:242, SEQ ID        NO:243, SEQ ID NO:244, SEQ ID NO:245, SEQ ID NO:192, SEQ ID        NO:381, SEQ ID NO:383, SEQ ID NO:385, SEQ ID NO:387, SEQ ID        NO:403, SEQ ID NO:405 or SEQ ID NO:407; or    -   (d) a complement of the nucleotide sequence of (a), (b) or (c),        wherein the complement and the nucleotide sequence consist of        the same number of nucleotides and are 100% complementary.

Preferably, an isolated polynucleotide encoding a delta-4 desaturasecomprises the sequence set forth in any of SEQ ID NO:214, SEQ ID NO:220,SEQ ID NO:236, SEQ ID NO:237, SEQ ID NO:238, SEQ ID NO:242, SEQ IDNO:243, SEQ ID NO:244, SEQ ID NO:245, SEQ ID NO:192, SEQ ID NO:381, SEQID NO:383, SEQ ID NO:385, SEQ ID NO:387, SEQ ID NO:403, SEQ ID NO:405,or SEQ ID NO:407.

The effect of truncating the Euglena anabaena delta-4 desaturase is thatenzymatic activity is increased when compared to enzymatic activity ofthe wildtype sequence. This result is unexpected and unforeseeable, asone of ordinary skill in the art would expect the activity of atruncated sequence to be no better and possibly less active than thewildtype sequence. Accordingly, the invention also provides a new methodfor deriving a delta-4 desaturase having higher activity than thewildtype sequence, the method comprising: a) providing a wild-typedelta-4 desaturase polypeptide isolated from Euglena anabena having abase-line delta-4 desaturase activity; and b) truncating the wild-typepolypeptide of (a) by about 1 to about 200 amino acids (a) to create atruncated mutant polypeptide having delta-4 desaturase activity that isincreased as compared with the baseline delta-4 desaturase activity.“Baseline” activity as used in this context is defined as the activityof the wildtype enzyme measured either in vivo or in vitro according tostandard enzymatic protocols as described herein.

In other embodiments, any of the enzymes (e.g., multizymes, DHAsynthases, C20 elongases, delta-4 desaturases, and/or any homologs)identified herein may be modified to generate new and/or improved PUFAbiosynthetic pathway enzymes. As is well known to those skilled in theart, in vitro mutagenesis and selection, chemical mutagenesis, “geneshuffling” methods or other means can be employed to obtain mutations ofnaturally occurring genes. Alternatively, multizymes may be synthesizedby domain swapping, wherein a functional domain from any enzyme may beexchanged with or added to a functional domain in an alternate enzyme tothereby result in a novel protein.

Methods for Production of Various Omega-3 and/or Omega-6 Fatty Acids

It is expected that introduction of chimeric genes encoding the DHAsynthases described herein (i.e., EgDHAsyn1, EgDHAsyn2, EaDHAsyn1,EaDHAsyn2 and EaDHAsyn3 or other mutant enzymes, codon-optimized enzymesor homologs thereof), under the control of the appropriate promoterswill result in increased production of DTA, DPAn-6, DPA and/or DHA inthe transformed host organism, respectively. As such, the presentinvention encompasses a method for the direct production of PUFAscomprising exposing a fatty acid substrate (i.e., EPA or DPA) to the DHAsynthase enzymes described herein (e.g., EgDHAsyn1, EgDHAsyn2,EaDHAsyn1, EaDHAsyn2 and EaDHAsyn3), such that the substrate isconverted to the desired fatty acid product (i.e., DHA).

More specifically, the present invention concerns a method fortransforming a host cell such that the host cell comprises in its genomea recombinant construct of the invention.

Examples of suitable host cells include, but are not limited to, plantsand yeast. Preferably, the plant cells are obtained from an oilseedplant such as soybean and the like and yeast cells are obtained fromoleaginous yeast such as Yarrowia sp.

Also within the scope of this invention is a method for producing atransformed plant or yeast comprising transforming a plant cell or ayeast cell with any of the polynucleotides of the invention andregenerating a plant from the transformed plant cell or growing thetransformed yeast cells.

More specifically, it is an object of the present invention to provide amethod for the production of DPAn-6 or DHA in a host cell (e.g., plants,oleaginous yeast), wherein the host cell comprises:

-   -   (i) an isolated nucleotide molecule encoding a polypeptide        having DHA synthase activity, wherein the polypeptide has at        least 80% amino acid identity, based on the Clustal V method of        alignment, when compared to an amino acid sequence as set forth        in SEQ ID NO:12, SEQ ID NO:22, SEQ ID NO:95, SEQ ID NO:96, or        SEQ ID NO:97; and,    -   (ii) a source of ARA or EPA;        wherein the host cell is grown under conditions such that the        polypeptide having DHA synthase activity is expressed and the        ARA is converted to DPAn-6 and/or the EPA is converted to DHA,        and wherein the DPAn-6 or DHA is optionally recovered.

In alternate embodiments, the present invention concerns a method forthe production of DTA or DPA in a host cell (e.g., plants, oleaginousyeast), wherein the host cell comprises:

-   -   (ii) an isolated nucleotide molecule encoding a polypeptide        having C20 elongase activity, wherein the polypeptide has at        least 80% amino acid identity, based on the Clustal V method of        alignment, when compared to an amino acid sequence as set forth        in SEQ ID NO:12, SEQ ID NO:22, SEQ ID NO:95, SEQ ID NO:96, SEQ        ID NO:97, SEQ ID NO:202, SEQ ID NO:204, SEQ ID NO:231, SEQ ID        NO:232, or SEQ ID NO:233; and,    -   (ii) a source of ARA or EPA;        wherein the host cell is grown under conditions such that the        polypeptide having C20 elongase activity is expressed and the        ARA is converted to DTA and/or the EPA is converted to DPA, and        wherein the DTA or DPA is optionally recovered.

Additionally, the invention provides a method for the production ofDPAn-6 or DHA, wherein the host cell comprises:

-   -   (i) an isolated nucleotide molecule encoding a polypeptide        having delta-4 desaturase activity, wherein the polypeptide has        at least 80% amino acid identity, based on the Clustal V method        of alignment, when compared to an amino acid sequence as set        forth in SEQ ID NO:12, SEQ ID NO:22, SEQ ID NO:95, SEQ ID NO:96,        SEQ ID NO:97, SEQ ID NO:215, SEQ ID NO:221, SEQ ID NO:239, SEQ        ID NO:240, SEQ ID NO:241, SEQ ID NO:246, SEQ ID NO:247, SEQ ID        NO:248, SEQ ID NO:249, SEQ ID NO:382, SEQ ID NO:384, SEQ ID        NO:386, SEQ ID NO:388, SEQ ID NO:404, SEQ ID NO:406, or SEQ ID        NO:408; and,    -   (ii) a source of DTA or DPA;        wherein the host cell is grown under conditions such that the        polypeptide having delta-4 desaturase activity is expressed and        the DTA is converted to DPAn-6 and/or the DPA is converted to        DHA, and wherein the DPAn-6 or DHA is optionally recovered.

The source of the substrate(s) ARA, DTA, EPA or DPA used in any of themethods above may be produced by the host either naturally ortransgenically, or may be provided exogenously.

Linking individual domains to form a multizyme could lead to a decreasein intermediate fatty acids. For instance, linking a C20 elongase with adelta-4 desaturase in a multizyme, such as DHA synthase, may lead to adecrease in the intermediate fatty acid DPA during production of DHA.Similarly, linking a delta-9 elongase with a delta-8 desaturase usingthe EgDHAsyn1 linker to form a multizyme as described herein may lead tothe production of DGLA and ETA with a decrease in EDA and ERAintermediates.

Alternatively, each multizyme gene including DHA synthase and theircorresponding enzyme products described herein can be used indirectlyfor the production of various omega-6 and omega-3 PUFAs, including e.g.,DTA, DPAn-6, DGLA, ETA, ARA, EPA, DPA and/or DHA (FIG. 1; see U.S. Pat.No. 7,238,482). Indirect production of omega-3/omega-6 PUFAs occurswherein the fatty acid substrate is converted indirectly into thedesired fatty acid product, via means of an intermediate step(s) orpathway intermediate(s). Thus, it is contemplated that the DHA synthasesdescribed herein (i.e., EgDHAsyn1, EgDHAsyn2, EaDHAsyn1, EaDHAsyn2 andEaDHAsyn3, or other mutant enzymes, codon-optimized enzymes or homologsthereof) may be expressed in conjunction with additional genes encodingenzymes of the PUFA biosynthetic pathway (e.g., delta-6 desaturases,C_(18/20) elongases, delta-17 desaturases, delta-8 desaturases, delta-15desaturases, delta-9 desaturases, delta-12 desaturases, C_(14/16)elongases, C_(16/18) elongases, delta-9 elongases, delta-5 desaturases,delta-4 desaturases, C_(20/22) elongases, DHA synthases) to result inhigher levels of production of longer-chain omega-3/omega-6 fatty acids(e.g., ARA, DTA, DPAn-6, EPA, DPA and/or DHA).

The specific genes included within a particular expression cassette willdepend on the host cell (and its PUFA profile and/or desaturase/elongaseprofile), the availability of substrate and the desired end product(s).

At times, it may be desirable to minimize by-product fatty acids. Therelative abundance of by-product fatty acids could be decreased bylinking individual pathway enzymes together with a linker to form amultizyme. For instance, the presence of sciadonic acid (SCI) and/orjuniperonic acid (JUP) [commonly found in the seed lipids of gymnosperms(Wolff et al., Lipids 35(1):1-22 (2000)), such as those in the Pinaceaefamily (pine)] might be considered by-product fatty acids of a delta-6desaturase/delta-6 elongase pathway or delta-9-elongase/delta-8desaturase pathway. Although these fatty acids are considered to havevarious health-enhancing properties themselves (Nakane et al., Biol.Pharm. Bull. 23: 758-761 (2000)), their presence as by-product fattyacids in an engineered PUFA pathway, such as in an oilseed crop, may notbe desirable depending on the application. Linking a delta-9 elongasetogether with a delta-8 desaturase using a linker to form a multizyme(DGLA and/or ETA synthase), for example, could result in increased fluxthrough these steps leading to reduced availability of the EDA/ERAintermediate fatty acids to delta-5 desaturase, and thus reducedconcentrations of SCI and JUP.

Occasionally, a delta-6 elongase may elongate fatty acids other than theintended fatty acid. For instance, delta-6 elongases generally convertGLA to DGLA but some delta-6 elongases may also convert unintendedsubstrates such as LA or ALA to EDA or ETrA, respectively. In a delta-6desaturase/delta-6 elongase pathway, EDA and ETrA would be considered“by-product fatty acids”. Addition of a delta-8 desaturase to a delta-6desaturase/delta-6 elongase pathway may provide a means to convert the“by-product fatty acids” EDA and ETrA back into the “intermediate fattyacids” DGLA and ETA, respectively.

In alternative embodiments, it may be useful to disrupt a hostorganism's native DHA synthase, C20 elongase, or delta-4 desaturase,based on the complete sequences described herein, the complement ofthose complete sequences, substantial portions of those sequences,codon-optimized desaturases derived therefrom, and those sequences thatare substantially homologous thereto.

Plant Expression Systems, Cassettes and Vectors, and Transformation

In one embodiment, this invention concerns a recombinant constructcomprising any one of the isolated polynucleotides of the inventionoperably linked to at least one regulatory sequence suitable forexpression in a host cell such as a plant. A promoter is a DNA sequencethat directs cellular machinery of a plant to produce RNA from thecontiguous coding sequence downstream (3′) of the promoter. The promoterregion influences the rate, developmental stage, and cell type in whichthe RNA transcript of the gene is made. The RNA transcript is processedto produce mRNA which serves as a template for translation of the RNAsequence into the amino acid sequence of the encoded polypeptide. The 5′non-translated leader sequence is a region of the mRNA upstream of theprotein coding region that may play a role in initiation and translationof the mRNA. The 3′ transcription termination/polyadenylation signal isa non-translated region downstream of the protein coding region thatfunctions in the plant cell to cause termination of the RNA transcriptand the addition of polyadenylate nucleotides to the 3′ end of the RNA.

The origin of the promoter chosen to drive expression of the multizymecoding sequence is not important as long as it has sufficienttranscriptional activity to accomplish the invention by expressingtranslatable mRNA for the desired nucleic acid fragments in the desiredhost tissue at the right time. Either heterologous or non-heterologous(i.e., endogenous) promoters can be used to practice the invention. Forexample, suitable promoters in plants include, but are not limited to:the alpha prime subunit of beta conglycinin promoter, the Kunitz trypsininhibitor 3 promoter, the annexin promoter, the glycinin Gy1 promoter,the beta subunit of beta conglycinin promoter, the P34/Gly Bd m 30Kpromoter, the albumin promoter, the Leg A1 promoter and the Leg A2promoter.

The annexin, or P34, promoter is described in PCT Publication No. WO2004/071178 (published Aug. 26, 2004). The level of activity of theannexin promoter is comparable to that of many known strong promoters,such as: (1) the CaMV 35S promoter (Atanassova et al., Plant Mol. Biol.37:275-285 (1998); Battraw and Hall, Plant Mol. Biol. 15:527-538 (1990);Holtorf et al., Plant Mol. Biol. 29:637-646 (1995); Jefferson et al.,EMBO J. 6:3901-3907 (1987); Wilmink et al., Plant Mol. Biol. 28:949-955(1995)); (2) the Arabidopsis oleosin promoters (Plant et al., Plant Mol.Biol. 25:193-205 (1994); L1, Texas A&M University Ph.D. dissertation,pp. 107-128 (1997)); (3) the Arabidopsis ubiquitin extension proteinpromoters (Callis et al., J. Biol. Chem. 265(21):12486-93 (1990)); (4) atomato ubiquitin gene promoter (Rollfinke et al., Gene. 211(2):267-76(1998)); (5) a soybean heat shock protein promoter (Schoffl et al., MolGen Genet. 217(2-3):246-53 (1989)); and, (6) a maize H3 histone genepromoter (Atanassova et al., Plant Mol. Biol. 37(2):275-85 (1989)).

Another useful feature of the annexin promoter is its expression profilein developing seeds. The annexin promoter is most active in developingseeds at early stages (before 10 days after pollination) and is largelyquiescent in later stages. The expression profile of the annexinpromoter is different from that of many seed-specific promoters, e.g.,seed storage protein promoters, which often provide highest activity inlater stages of development (Chen et al., Dev. Genet. 10:112-122 (1989);Ellerstrom et al., Plant Mol. Biol. 32:1019-1027 (1996); Keddie et al.,Plant Mol. Biol. 24:327-340 (1994); Plant et al., (supra); Li, (supra)).The annexin promoter has a more conventional expression profile butremains distinct from other known seed specific promoters. Thus, theannexin promoter will be a very attractive candidate whenoverexpression, or suppression, of a gene in embryos is desired at anearly developing stage. For example, it may be desirable to overexpressa gene regulating early embryo development or a gene involved in themetabolism prior to seed maturation.

Following identification of an appropriate promoter suitable forexpression of a specific DHA synthase coding sequence, the promoter isthen operably linked in a sense orientation using conventional meanswell known to those skilled in the art.

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook, J.et al., In Molecular Cloning: A Laboratory Manual; 2^(nd) ed.; ColdSpring Harbor Laboratory Press: Cold Spring Harbor, N.Y., 1989(hereinafter “Sambrook et al., 1989”) or Ausubel, F. M., Brent, R.,Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. and Struhl,K., Eds.; In Current Protocols in Molecular Biology; John Wiley andSons: New York, 1990 (hereinafter “Ausubel et al., 1990”). For example,a fusion gene can be constructed by linking at least two DNA fragmentsin frame so as not to introduce a stop codon (in-frame fusion). Theresulting fusion gene will be such that each DNA fragment encodes for atleast one independent and separable enzymatic activity.

Once the recombinant construct has been made, it may then be introducedinto a plant cell of choice by methods well known to those of ordinaryskill in the art (e.g., transfection, transformation andelectroporation). Oilseed plant cells are the preferred plant cells. Thetransformed plant cell is then cultured and regenerated under suitableconditions permitting expression of the long-chain PUFA which is thenoptionally recovered and purified.

The recombinant constructs of the invention may be introduced into oneplant cell; or, alternatively, each construct may be introduced intoseparate plant cells.

Expression in a plant cell may be accomplished in a transient or stablefashion as is described above.

The desired long-chain PUFAs can be expressed in seed. Also within thescope of this invention are seeds or plant parts obtained from suchtransformed plants.

Plant parts include differentiated and undifferentiated tissuesincluding, but not limited to the following: roots, stems, shoots,leaves, pollen, seeds, tumor tissue and various forms of cells andculture (e.g., single cells, protoplasts, embryos and callus tissue).The plant tissue may be in plant or in a plant organ, tissue or cellculture.

The term “plant organ” refers to plant tissue or a group of tissues thatconstitute a morphologically and functionally distinct part of a plant.The term “genome” refers to the following: (1) the entire complement ofgenetic material (genes and non-coding sequences) that is present ineach cell of an organism, or virus or organelle; and/or (2) a completeset of chromosomes inherited as a (haploid) unit from one parent.

Thus, this invention also concerns a method for transforming a cell,comprising transforming a cell with the recombinant construct of theinvention and selecting those cells transformed with the recombinantconstructs described in the claims.

Also of interest is a method for producing a transformed plantcomprising transforming a plant cell with the polynucleotides of theinstant invention and regenerating a plant from the transformed plantcell.

Methods for transforming dicots (primarily by use of Agrobacteriumtumefaciens) and obtaining transgenic plants have been published, amongothers, for: cotton (U.S. Pat. No. 5,004,863; U.S. Pat. No. 5,159,135);soybean (U.S. Pat. No. 5,569,834; U.S. Pat. No. 5,416,011); Brassica(U.S. Pat. No. 5,463,174); peanut (Cheng et al. Plant Cell Rep.15:653-657 (1996); McKently et al. Plant Cell Rep. 14:699-703 (1995));papaya (Ling, K. et al. Bio/technology 9:752-758 (1991)); and pea (Grantet al. Plant Cell Rep. 15:254-258 (1995)). For a review of othercommonly used methods of plant transformation see Newell, C. A. (Mol.Biotechnol. 16:53-65 (2000)). One of these methods of transformationuses Agrobacterium rhizogenes (Tepfler, M. and Casse-Delbart, F.Microbiol. Sci. 4:24-28 (1987)). Transformation of soybeans using directdelivery of DNA has been published using PEG fusion (PCT Publication No.WO 92/17598), electroporation (Chowrira, G. M. et al., Mol. Biotechnol.3:17-23 (1995); Christou, P. et al., Proc. Natl. Acad. Sci. U.S.A.84:3962-3966 (1987)), microinjection and particle bombardement (McCabe,D. E. et. al., Bio/Technology 6:923 (1988); Christou et al., PlantPhysiol. 87:671-674 (1988)).

There are a variety of methods for the regeneration of plants from planttissue. The particular method of regeneration will depend on thestarting plant tissue and the particular plant species to beregenerated. The regeneration, development and cultivation of plantsfrom single plant protoplast transformants or from various transformedexplants is well known in the art (Weissbach and Weissbach, In: Methodsfor Plant Molecular Biology, (Eds.), Academic: San Diego, Calif.(1988)). This regeneration and growth process typically includes thesteps of selection of transformed cells and culturing thoseindividualized cells through the usual stages of embryonic developmentthrough the rooted plantlet stage. Transgenic embryos and seeds aresimilarly regenerated. The resulting transgenic rooted shoots arethereafter planted in an appropriate plant growth medium such as soil.Preferably, the regenerated plants are self-pollinated to providehomozygous transgenic plants. Otherwise, pollen obtained from theregenerated plants is crossed to seed-grown plants of agronomicallyimportant lines. Conversely, pollen from plants of these important linesis used to pollinate regenerated plants. A transgenic plant of thepresent invention containing a desired polypeptide is cultivated usingmethods well known to one skilled in the art.

In addition to the above discussed procedures, practitioners arefamiliar with the standard resource materials which describe specificconditions and procedures for: the construction, manipulation andisolation of macromolecules (e.g., DNA molecules, plasmids, etc.); thegeneration of recombinant DNA fragments and recombinant expressionconstructs; and, the screening and isolating of clones. See, forexample: Sambrook et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor: NY (1989); Maliga et al., Methods in Plant MolecularBiology, Cold Spring Harbor: NY (1995); Birren et al., Genome Analysis:Detecting Genes, Vol. 1, Cold Spring Harbor: NY (1998); Birren et al.,Genome Analysis: Analyzing DNA, Vol. 2, Cold Spring Harbor: NY (1998);Plant Molecular Biology: A Laboratory Manual, eds. Clark, Springer: NY(1997).

Examples of oilseed plants include, but are not limited to: soybean,Brassica species, sunflower, maize, cotton, flax and safflower.

Examples of PUFAs having at least twenty carbon atoms and four or morecarbon-carbon double bonds include, but are not limited to, omega-3fatty acids such as EPA, DPA, and DHA. Seeds obtained from such plantsare also within the scope of this invention as well as oil obtained fromsuch seeds.

Thus, the present invention also concerns a method for altering thefatty acid profile of an oilseed plant comprising:

a) transforming an oilseed plant cell with the recombinant construct ofclaim of the invention; and

b) regenerating a plant from the transformed oilseed plant cell step(a), wherein the plant has an altered fatty acid profile.

Microbial Expression Systems, Cassettes and Vectors

The DHA synthase genes and gene products described herein (i.e.,EgDHAsyn1, EgDHAsyn2, EaDHAsyn1, EaDHAsyn2 and EaDHAsyn3, or othermutant enzymes, codon-optimized enzymes or homologs thereof) may also beproduced in heterologous microbial host cells, particularly in the cellsof oleaginous yeasts (e.g., Yarrowia lipolytica).

Microbial expression systems and expression vectors containingregulatory sequences that direct high level expression of foreignproteins are well known to those skilled in the art. Any of these couldbe used to construct chimeric genes for production of any of the geneproducts of the instant sequences. These chimeric genes could then beintroduced into appropriate microorganisms via transformation to providehigh-level expression of the encoded enzymes.

Vectors useful for the transformation of suitable microbial host cellsare well known in the art. The specific choice of sequences present inthe construct is dependent upon the desired expression products (supra),the nature of the host cell and the proposed means of separatingtransformed cells versus non-transformed cells. Typically, however, thevector contains at least one expression cassette, a selectable markerand sequences allowing autonomous replication or chromosomalintegration. Suitable expression cassettes comprise a region 5′ of thegene that controls transcriptional initiation (e.g., a promoter), thegene coding sequence, and a region 3′ of the DNA fragment that controlstranscriptional termination (i.e., a terminator). It is most preferredwhen both control regions are derived from genes from the transformedmicrobial host cell, although it is to be understood that such controlregions need not be derived from the genes native to the specificspecies chosen as a production host.

Initiation control regions or promoters which are useful to driveexpression of the instant multizymes, such as DHA synthase or individualdomain ORFs, in the desired microbial host cell are numerous andfamiliar to those skilled in the art. Virtually any promoter capable ofdirecting expression of these genes in the selected host cell issuitable for the present invention. Expression in a microbial host cellcan be accomplished in a transient or stable fashion. Transientexpression can be accomplished by inducing the activity of a regulatablepromoter operably linked to the gene of interest. Stable expression canbe achieved by the use of a constitutive promoter operably linked to thegene of interest. As an example, when the host cell is yeast,transcriptional and translational regions functional in yeast cells areprovided, particularly from the host species (e.g., see U.S. Pat. No.7,238,482 and PCT Publication No. WO 2006/052870 for preferredtranscriptional initiation regulatory regions for use in Yarrowialipolytica). Any one of a number of regulatory sequences can be used,depending upon whether constitutive or induced transcription is desired,the efficiency of the promoter in expressing the ORF of interest, theease of construction and the like.

Nucleotide sequences surrounding the translational initiation codon‘ATG’ have been found to affect expression in yeast cells. If thedesired polypeptide is poorly expressed in yeast, the nucleotidesequences of exogenous genes can be modified to include an efficientyeast translation initiation sequence to obtain optimal gene expression.For expression in yeast, this can be done by site-directed mutagenesisof an inefficiently expressed gene by fusing it in-frame to anendogenous yeast gene, preferably a highly expressed gene.Alternatively, one can determine the consensus translation initiationsequence in the host and engineer this sequence into heterologous genesfor their optimal expression in the host of interest.

The termination region can be derived from the 3′ region of the genefrom which the initiation region was obtained or from a different gene.A large number of termination regions are known and functionsatisfactorily in a variety of hosts (when utilized both in the same anddifferent genera and species from where they were derived). Thetermination region usually is selected more as a matter of conveniencerather than because of any particular property. Termination controlregions may also be derived from various genes native to the preferredhosts. In alternate embodiments, the 3′-region can also be synthetic, asone of skill in the art can utilize available information to design andsynthesize a 3′-region sequence that functions as a transcriptionterminator. Optionally, a termination site may be unnecessary; however,it is most preferred if included.

As one of skill in the art is aware, merely inserting a gene into acloning vector does not ensure that it will be successfully expressed atthe level needed. In response to the need for a high expression rate,many specialized expression vectors have been created by manipulating anumber of different genetic elements that control aspects oftranscription, translation, protein stability, oxygen limitation andsecretion from the microbial host cell. More specifically, some of themolecular features that have been manipulated to control gene expressioninclude: the nature of the relevant transcriptional promoter andterminator sequences; the number of copies of the cloned gene; whetherthe gene is plasmid-borne or integrated into the genome of the hostcell; the final cellular location of the synthesized foreign protein;the efficiency of translation and correct folding of the protein in thehost organism; the intrinsic stability of the mRNA and protein of thecloned gene within the host cell; and the codon usage within the clonedgene, such that its frequency approaches the frequency of preferredcodon usage of the host cell. Each type of modification is encompassedin the present invention, as means to further optimize expression of theDHA synthases described herein.

Transformation of Microbial Host Cells

Once a cassette that is suitable for expression in an appropriate hostcell has been obtained (e.g., a chimeric gene comprising a promoter, ORFand terminator), it is placed in a plasmid vector capable of autonomousreplication in a host cell, or is directly integrated into the genome ofthe host cell. Integration of expression cassettes can occur randomlywithin the host genome or can be targeted through the use of constructscontaining regions of homology with the host genome sufficient to targetrecombination within the host locus. Where constructs are targeted to anendogenous locus, all or some of the transcriptional and translationalregulatory regions can be provided by the endogenous locus.

Where two or more genes are expressed from separate replicating vectors,it is desirable that each vector has a different means of selection andshould lack homology to the other construct(s) to maintain stableexpression and prevent reassortment of elements among constructs.Judicious choice of regulatory regions, selection means and method ofpropagation of the introduced construct(s) can be experimentallydetermined so that all introduced genes are expressed at the necessarylevels to provide for synthesis of the desired products.

Constructs comprising the gene(s) of interest may be introduced into amicrobial host cell by any standard technique. These techniques includetransformation (e.g., lithium acetate transformation [Methods inEnzymology, 194:186-187 (1991)]), protoplast fusion, biolistic impact,electroporation, microinjection, or any other method that introduces thegene(s) of interest into the host cell.

For convenience, a host cell that has been manipulated by any method totake up a DNA sequence (e.g., an expression cassette) will be referredto as “transformed” or “recombinant” herein. The transformed host willhave at least one copy of the expression construct and may have two ormore, depending upon whether the expression cassette is integrated intothe genome or is present on an extrachromosomal element having multiplecopy numbers.

The transformed host cell can be identified by various selectiontechniques, as described in U.S. Pat. Nos. 7,238,482 and 7,259,255 andPCT Publication No. WO 2006/052870.

Following transformation, substrates suitable for the instant DHAsynthases (and, optionally other PUFA enzymes that are co-expressedwithin the host cell) may be produced by the host either naturally ortransgenically, or may be provided exogenously.

Preferred Microbial Hosts for Recombinant Expression

Microbial host cells for expression of the instant genes and nucleicacid fragments may include hosts that grow on a variety of feedstocks,including simple or complex carbohydrates, fatty acids, organic acids,oils and alcohols, and/or hydrocarbons over a wide range of temperatureand pH values. Based on the needs of the Applicants' Assignee, the genesdescribed in the instant invention will be expressed in an oleaginousyeast (and in particular Yarrowia lipolytica); however, it iscontemplated that because transcription, translation and the proteinbiosynthetic apparatus are highly conserved, any bacteria, yeast, algae,euglenoid and/or fungus will be a suitable microbial host for expressionof the present nucleic acid fragments.

Preferred microbial hosts, however, are oleaginous organisms, such asoleaginous yeasts. These organisms are naturally capable of oilsynthesis and accumulation, wherein the oil can comprise greater thanabout 25% of the cellular dry weight, more preferably greater than about30% of the cellular dry weight, and most preferably greater than about40% of the cellular dry weight. Genera typically identified asoleaginous yeast include, but are not limited to: Yarrowia, Candida,Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces.More specifically, illustrative oil-synthesizing yeasts include:Rhodosporidium toruloides, Lipomyces starkeyii, L. lipoferus, Candidarevkaufi, C. pulcherrima, C. tropicalis, C. utilis, Trichosporonpullans, T. cutaneum, Rhodotorula glutinus, R. graminis, and Yarrowialipolytica (formerly classified as Candida lipolytica).

Most preferred is the oleaginous yeast Yarrowia lipolytica; and, in afurther embodiment, most preferred are the Y. lipolytica strainsdesignated as ATCC #20362, ATCC #8862, ATCC #18944, ATCC #76982 and/orLGAM S(7)1 (Papanikolaou S., and Aggelis G., Bioresour. Technol.82(1):43-9 (2002)).

Historically, various strains of Y. lipolytica have been used for themanufacture and production of: isocitrate lyase; lipases;polyhydroxyalkanoates; citric acid; erythritol; 2-oxoglutaric acid;gamma-decalactone; gamma-dodecalatone; and pyruvic acid. Specificteachings applicable for transformation of oleaginous yeasts (i.e.,Yarrowia lipolytica) include U.S. Pat. No. 4,880,741 and U.S. Pat. No.5,071,764 and Chen, D. C. et al. (Appl. Microbiol. Biotechnol.,48(2):232-235 (1997)). Specific teachings applicable for engineeringARA, EPA and DHA production in Y. lipolytica are provided in U.S. patentapplication Ser. No. 11/264,784 (PCT Publication No. WO 2006/055322),U.S. patent application Ser. No. 11/265,761 (PCT Publication No. WO2006/052870) and U.S. patent application Ser. No. 11/264,737 (PCTPublication No. WO 2006/052871), respectively.

Detailed means for the synthesis and transformation of expressionvectors comprising C20 elongases and delta-4 desaturases in oleaginousyeast (i.e., Yarrowia lipolytica) are provided in PCT Publication No. WO2006/052871. The preferred method of expressing genes in Yarrowialipolytica is by integration of linear DNA into the genome of the host.Integration into multiple locations within the genome can beparticularly useful when high level expression of genes are desired[e.g., in the Ura3 locus (GenBank Accession No. AJ306421), the Leu2 genelocus (GenBank Accession No. AF260230), the Lys5 gene locus (GenBankAccession No. M34929), the Aco2 gene locus (GenBank Accession No.AJ001300), the Pox3 gene locus (Pox3: GenBank Accession No.XP_(—)503244; or, Aco3: GenBank Accession No. AJ001301), the delta-12desaturase gene locus (U.S. Pat. No. 7,214,491), the Lip1 gene locus(GenBank Accession No. Z50020), the Lip2 gene locus (GenBank AccessionNo. AJ012632), and/or the Pex10 gene locus (GenBank Accession No.CAG81606)].

Termination regions useful in the disclosure herein for Yarrowiaexpression vectors include, for example: ˜100 by of the 3′ region of theYarrowia lipolytica extracellular protease (XPR; GenBank Accession No.M17741); the acyl-CoA oxidase (Aco3: GenBank Accession No. AJ001301 andNo. CAA04661; Pox3: GenBank Accession No. XP_(—)503244) terminators; thePex20 (GenBank Accession No. AF054613) terminator; the Pex16 (GenBankAccession No. U75433) terminator; the Lip1 (GenBank Accession No.Z50020) terminator; the Lip2 (GenBank Accession No. AJ012632)terminator; and the 3-oxoacyl-CoA thiolase (OCT; GenBank Accession No.X69988) terminator.

Preferred selection methods for use in Yarrowia lipolytica areresistance to kanamycin, hygromycin, and the amino glycoside G418, aswell as the ability to grow on media lacking uracil, leucine, lysine,tryptophan or histidine. In alternate embodiments, 5-fluoroorotic acid(5-fluorouracil-6-carboxylic acid monohydrate; “5-FOA”) is used forselection of yeast Ura⁻ mutants. The compound is toxic to yeast cellsthat possess a functioning URA3 gene encoding orotidine 5′-monophosphatedecarboxylase (OMP decarboxylase); thus, based on this toxicity, 5-FOAis especially useful for the selection and identification of Ura⁻ mutantyeast strains (Bartel, P. L. and Fields, S., Yeast 2-Hybrid System,Oxford University: New York, v. 7, pp 109-147, 1997; see also PCTPublication No. WO 2006/052870 for 5-FOA use in Yarrowia). Morespecifically, one can first knockout the native Ura3 gene to produce astrain having a Ura− phenotype, wherein selection occurs based on 5-FOAresistance. Then, a cluster of multiple chimeric genes and a new Ura3gene can be integrated into a different locus of the Yarrowia genome toproduce a new strain having a Ura+ phenotype. Subsequent integrationproduces a new Ura3-strain (again identified using 5-FOA selection),when the introduced Ura3 gene is knocked out. Thus, the Ura3 gene (incombination with 5-FOA selection) can be used as a selection marker inmultiple rounds of transformation, thereby readily permitting geneticmodifications to be integrated into the Yarrowia genome in a facilemanner.

Other preferred microbial hosts include oleaginous bacteria, algae,euglenoids, and other fungi; and, within this broad group of microbialhosts, of particular interest are microorganisms that synthesizeomega-3/omega-6 fatty acids (or those that can be genetically engineeredfor this purpose [e.g., other yeast such as Saccharomyces cerevisiae]).Thus, for example, transformation of Mortierella alpina (which iscommercially used for production of ARA) with any of the instant DHAsynthase genes under the control of inducible or regulated promoterscould yield a transformant organism capable of synthesizing increasedquantities of PUFAs. The method of transformation of M. alpina isdescribed by Mackenzie et al. (Appl. Environ. Microbiol., 66:4655(2000)). Similarly, methods for transformation of Thraustochytrialesmicroorganisms are disclosed in U.S. Pat. No. 7,001,772.

Substrate feeding may be required.

Irrespective of the host selected for expression of the multizymes (e.g.DHA synthases), multiple transformants must be screened in order toobtain a strain displaying the desired expression level and pattern.Such screening may be accomplished by Southern analysis of DNA blots(Southern, J. Mol. Biol., 98:503 (1975)), Northern analysis of mRNAexpression (Kroczek, J. Chromatogr. Biomed. Appl., 618(1-2):133-145(1993)), Western and/or Elisa analyses of protein expression, phenotypicanalysis, or GC analysis of the PUFA products.

Of course, since naturally produced PUFAs in oleaginous yeast arelimited to 18:2 fatty acids (i.e., LA), and less commonly, 18:3 fattyacids (i.e., ALA), in more preferred embodiments of the presentinvention, the oleaginous yeast will be genetically engineered toexpress multiple enzymes necessary for long-chain PUFA biosynthesis(thereby enabling production of e.g., ARA, EPA, DPA and DHA), inaddition to the multizymes described herein.

In particularly preferred embodiments, the at least one additionalrecombinant DNA construct encode a DGLA synthase, such that themultizyme has both delta-9 elongase activity and delta-8 desaturaseactivity. In some embodiments the delta-9 elongase can be isolated orderived from Isochrysis galbana (GenBank Accession No. AF390174; IgD9eor IgD9eS) or the delta-9 elongase can be isolated or derived fromEuglena gracilis or Euglena anabaena. For example, see the DGLAsynthases set forth as SEQ ID NO:441, SEQ ID NO:447, SEQ ID NO:454, SEQID NO:461, SEQ ID NO:464 and SEQ ID NO:471.

Metabolic Engineering of Omega-3 and/or Omega-6 Fatty Acid Biosynthesisin Microbes

Methods for manipulating biochemical pathways are well known to thoseskilled in the art; and, it is expected that numerous manipulations willbe possible to maximize omega-3 and/or omega-6 fatty acid biosynthesisin oleaginous yeasts, and particularly, in Yarrowia lipolytica. Thismanipulation may require metabolic engineering directly within the PUFAbiosynthetic pathway or additional manipulation of pathways thatcontribute carbon to the PUFA biosynthetic pathway. Methods useful forup-regulating desirable biochemical pathways and down-regulatingundesirable biochemical pathways are well known to those skilled in theart.

For example, biochemical pathways competing with the omega-3 and/oromega-6 fatty acid biosynthetic pathways for energy or carbon, or nativePUFA biosynthetic pathway enzymes that interfere with production of aparticular PUFA end-product, may be eliminated by gene disruption ordown-regulated by other means (e.g., antisense mRNA).

Detailed discussion of manipulations within the PUFA biosyntheticpathway as a means to increase ARA, EPA or DHA (and associatedtechniques thereof) are presented in PCT Publication Nos. WO 2006/055322[U.S. Patent Publication No. 2006-0094092-A1], PCT Publication No. WO2006/052870 [U.S. Patent Publication No. 2006-0115881-A1] and PCTPublication No. WO 2006/052871 [U.S. Patent Publication No.2006-0110806-A1], respectively, as are desirable manipulations in theTAG biosynthetic pathway and the TAG degradation pathway (and associatedtechniques thereof).

Within the context of the present invention, it may be useful tomodulate the expression of the fatty acid biosynthetic pathway by anyone of the strategies described above. For example, the presentinvention provides methods whereby genes encoding key enzymes in thePUFA biosynthetic pathway are introduced into oleaginous yeasts for theproduction of omega-3 and/or omega-6 fatty acids. It will beparticularly useful to express the instant DHA synthase genes inoleaginous yeasts that do not naturally possess omega-3 and/or omega-6fatty acid biosynthetic pathways and coordinate the expression of thesegenes, to maximize production of preferred PUFA products using variousmeans for metabolic engineering of the host organism.

Microbial Fermentation Processes for PUFA Production

The transformed host cell is grown under conditions that optimizeexpression of chimeric genes and produce the greatest and mosteconomical yield of desired PUFAs. In general, media conditions that maybe optimized include the type and amount of carbon source, the type andamount of nitrogen source, the carbon-to-nitrogen ratio, the amount ofdifferent mineral ions, the oxygen level, growth temperature, pH, lengthof the biomass production phase, length of the oil accumulation phaseand the time and method of cell harvest. Yarrowia lipolytica aregenerally grown in complex media (e.g., yeast extract-peptone-dextrosebroth (YPD)) or a defined minimal media that lacks a component necessaryfor growth and thereby forces selection of the desired expressioncassettes (e.g., Yeast Nitrogen Base (DIFCO Laboratories, Detroit,Mich.)).

Fermentation media in the present invention must contain a suitablecarbon source. Suitable carbon sources are taught in U.S. Pat. No.7,238,482. Although it is contemplated that the source of carbonutilized in the present invention may encompass a wide variety ofcarbon-containing sources, preferred carbon sources are sugars,glycerol, and/or fatty acids. Most preferred is glucose and/or fattyacids containing between 10-22 carbons.

Nitrogen may be supplied from an inorganic (e.g., (NH₄)₂SO₄) or organic(e.g., urea or glutamate) source. In addition to appropriate carbon andnitrogen sources, the fermentation media must also contain suitableminerals, salts, cofactors, buffers, vitamins and other components knownto those skilled in the art suitable for the growth of the oleaginoushost and promotion of the enzymatic pathways necessary for PUFAproduction. Particular attention is given to several metal ions (e.g.,Fe⁺², Cu⁺², Mn⁺², Co⁺², Zn⁺², Mg⁺²) that promote synthesis of lipids andPUFAs (Nakahara, T. et al., Ind. Appl. Single Cell Oils, D. J. Kyle andR. Colin, eds. pp 61-97 (1992)).

Preferred growth media in the present invention are common commerciallyprepared media, such as Yeast Nitrogen Base (DIFCO Laboratories,Detroit, Mich.). Other defined or synthetic growth media may also beused and the appropriate medium for growth of the transformant hostcells will be known by one skilled in the art of microbiology orfermentation science. A suitable pH range for the fermentation istypically between about pH 4.0 to pH 8.0, wherein pH 5.5 to pH 7.5 ispreferred as the range for the initial growth conditions. Thefermentation may be conducted under aerobic or anaerobic conditions,wherein microaerobic conditions are preferred.

Typically, accumulation of high levels of PUFAs in oleaginous yeastcells requires a two-stage process, since the metabolic state must be“balanced” between growth and synthesis/storage of fats. Thus, mostpreferably, a two-stage fermentation process is necessary for theproduction of PUFAs in oleaginous yeast (e.g., Yarrowia lipolytica).This approach is described in U.S. Pat. No. 7,238,482, as are varioussuitable fermentation process designs (i.e., batch, fed-batch andcontinuous) and considerations during growth.

Purification and Processing of PUFA Oils

PUFAs may be found in the host microorganisms and plants as free fattyacids or in esterified forms such as acylglycerols, phospholipids,sulfolipids, or glycolipids, and may be extracted from the host cellsthrough a variety of means well-known in the art. One review ofextraction techniques, quality analysis, and acceptability standards foryeast lipids is that of Z. Jacobs (Critical Reviews in Biotechnology,12(5/6):463-491 (1992)). A brief review of downstream processing is alsoavailable by A. Singh and O. Ward (Adv. Appl. Microbiol., 45:271-312(1997)).

In general, means for the purification of PUFAs may include extraction(e.g., U.S. Pat. No. 6,797,303 and U.S. Pat. No. 5,648,564) with organicsolvents, sonication, supercritical fluid extraction (e.g., using carbondioxide), saponification and physical means such as presses, orcombinations thereof. One is referred to the teachings of U.S. Pat. No.7,238,482 for additional details. Methods of isolating seed oils arewell known in the art: (Young et al., Processing of Fats and Oils, InThe Lipid Handbook, Gunstone et al., eds., Chapter 5 pp 253-257; Chapman& Hall: London (1994)). For example, soybean oil is produced using aseries of steps involving the extraction and purification of an edibleoil product from the oil-bearing seed. Soybean oils and soybeanbyproducts are produced using the generalized steps shown in Table 7.

TABLE 7 Generalized Steps for Soybean Oil and Byproduct ProductionProcess Impurities Removed and/or Step Process By-Products Obtained # 1soybean seed # 2 oil extraction meal # 3 Degumming lecithin # 4 alkalior physical gums, free fatty acids, refining pigments # 5 water washingsoap # 6 Bleaching color, soap, metal # 7 (hydrogenation) # 8(winterization) stearine # 9 Deodorization free fatty acids,tocopherols, sterols, volatiles # 10  oil products

More specifically, soybean seeds are cleaned, tempered, dehulled, andflaked, thereby increasing the efficiency of oil extraction. Oilextraction is usually accomplished by solvent (e.g., hexane) extractionbut can also be achieved by a combination of physical pressure and/orsolvent extraction. The resulting oil is called crude oil. The crude oilmay be degummed by hydrating phospholipids and other polar and neutrallipid complexes that facilitate their separation from the nonhydrating,triglyceride fraction (soybean oil). The resulting lecithin gums may befurther processed to make commercially important lecithin products usedin a variety of food and industrial products as emulsification andrelease (i.e., antisticking) agents. Degummed oil may be further refinedfor the removal of impurities (primarily free fatty acids, pigments andresidual gums). Refining is accomplished by the addition of a causticagent that reacts with free fatty acid to form soap and hydratesphosphatides and proteins in the crude oil. Water is used to wash outtraces of soap formed during refining. The soapstock byproduct may beused directly in animal feeds or acidulated to recover the free fattyacids. Color is removed through adsorption with a bleaching earth thatremoves most of the chlorophyll and carotenoid compounds. The refinedoil can be hydrogenated, thereby resulting in fats with various meltingproperties and textures. Winterization (fractionation) may be used toremove stearine from the hydrogenated oil through crystallization undercarefully controlled cooling conditions. Deodorization (principally viasteam distillation under vacuum) is the last step and is designed toremove compounds which impart odor or flavor to the oil. Other valuablebyproducts such as tocopherols and sterols may be removed during thedeodorization process. Deodorized distillate containing these byproductsmay be sold for production of natural vitamin E and other high-valuepharmaceutical products. Refined, bleached, (hydrogenated, fractionated)and deodorized oils and fats may be packaged and sold directly orfurther processed into more specialized products. A more detailedreference to soybean seed processing, soybean oil production, andbyproduct utilization can be found in Erickson, Practical Handbook ofSoybean Processing and Utilization, The American Oil Chemists' Societyand United Soybean Board (1995). Soybean oil is liquid at roomtemperature because it is relatively low in saturated fatty acids whencompared with oils such as coconut, palm, palm kernel, and cocoa butter.

Plant and microbial oils containing PUFAs that have been refined and/orpurified can be hydrogenated, thereby resulting in fats with variousmelting properties and textures. Many processed fats (including spreads,confectionary fats, hard butters, margarines, baking shortenings, etc.)require varying degrees of solidity at room temperature and can only beproduced through alteration of the source oil's physical properties.This is most commonly achieved through catalytic hydrogenation.

Hydrogenation is a chemical reaction in which hydrogen is added to theunsaturated fatty acid double bonds with the aid of a catalyst such asnickel. For example, high oleic soybean oil contains unsaturated oleic,linoleic, and linolenic fatty acids, and each of these can behydrogenated. Hydrogenation has two primary effects. First, theoxidative stability of the oil is increased as a result of the reductionof the unsaturated fatty acid content. Second, the physical propertiesof the oil are changed because the fatty acid modifications increase themelting point resulting in a semi-liquid or solid fat at roomtemperature.

There are many variables which affect the hydrogenation reaction, whichin turn alter the composition of the final product. Operating conditionsincluding pressure, temperature, catalyst type and concentration,agitation, and reactor design are among the more important parametersthat can be controlled. Selective hydrogenation conditions can be usedto hydrogenate the more unsaturated fatty acids in preference to theless unsaturated ones. Very light or brush hydrogenation is oftenemployed to increase stability of liquid oils. Further hydrogenationconverts a liquid oil to a physically solid fat. The degree ofhydrogenation depends on the desired performance and meltingcharacteristics designed for the particular end product. Liquidshortenings (used in the manufacture of baking products, solid fats andshortenings used for commercial frying and roasting operations) and basestocks for margarine manufacture are among the myriad of possible oiland fat products achieved through hydrogenation. A more detaileddescription of hydrogenation and hydrogenated products can be found inPatterson, H. B. W., Hydrogenation of Fats and Oils: Theory andPractice. The American Oil Chemists'Society (1994).

Hydrogenated oils have become somewhat controversial due to the presenceof trans-fatty acid isomers that result from the hydrogenation process.Ingestion of large amounts of trans-isomers has been linked withdetrimental health effects including increased ratios of low density tohigh density lipoproteins in the blood plasma and increased risk ofcoronary heart disease.

PUFA-Containing Oils for Use in Foodstuffs, Health Food Products,Pharmaceuticals and Animal Feeds

The market place currently supports a large variety of food and feedproducts, incorporating omega-3 and/or omega-6 fatty acids (particularlye.g., ALA, GLA, ARA, EPA, DPA and DHA). It is contemplated that thePUFA-comprising plant/seed oils, altered seeds, and microbial biomassand/or oils of the invention will function in food and feed products toimpart the health benefits of current formulations. Compared to othervegetable oils, the oils of the invention are believed to functionsimilarly to other oils in food applications from a physical standpoint(for example, partially hydrogenated oils such as soybean oil are widelyused as ingredients for soft spreads, margarine and shortenings forbaking and frying).

Plant/seed oils, altered seeds, and microbial biomass and/or oilscontaining omega-3 and/or omega-6 fatty acids will be suitable for usein a variety of food and feed products including, but not limited to:food analogs, meat products, cereal products, baked foods, snack foodsand dairy products.

Additionally, the present plant/seed oils, altered seeds, and microbialbiomass and/or oils may be used in formulations to impart health benefitin medical foods including medical nutritionals, dietary supplements,infant formula as well as pharmaceutical products. One of skill in theart of food processing and food formulation will understand how theamount and composition of the plant and microbial oils may be added tothe food or feed product. Such an amount will be referred to herein asan “effective” amount and will depend on the food or feed product, thediet that the product is intended to supplement or the medical conditionthat the medical food or medical nutritional is intended to correct ortreat.

Food analogs can be made using processes well known to those skilled inthe art. There can be mentioned meat analogs, cheese analogs, milkanalogs and the like. Meat analogs made from soybeans contain soyprotein or tofu and other ingredients mixed together to simulate variouskinds of meats. These meat alternatives are sold as frozen, canned ordried foods. Usually, they can be used the same way as the foods theyreplace. Meat alternatives made from soybeans are excellent sources ofprotein, iron and B vitamins. Examples of meat analogs include, but arenot limited to: ham analogs, sausage analogs, bacon analogs, and thelike.

Food analogs can be classified as imitation or substitutes depending ontheir functional and compositional characteristics. For example, animitation cheese need only resemble the cheese it is designed toreplace. However, a product can generally be called a substitute cheeseonly if it is nutritionally equivalent to the cheese it is replacing andmeets the minimum compositional requirements for that cheese. Thus,substitute cheese will often have higher protein levels than imitationcheeses and be fortified with vitamins and minerals.

Milk analogs or nondairy food products include, but are not limited to,imitation milks and nondairy frozen desserts (e.g., those made fromsoybeans and/or soy protein products).

Meat products encompass a broad variety of products. In the UnitedStates “meat” includes “red meats” produced from cattle, hogs and sheep.In addition to the red meats there are poultry items which includechickens, turkeys, geese, guineas, ducks and the fish and shellfish.There is a wide assortment of seasoned and processed meat products:fresh, cured and fried, and cured and cooked. Sausages and hot dogs areexamples of processed meat products. Thus, the term “meat products” asused herein includes, but is not limited to, processed meat products.

A cereal food product is a food product derived from the processing of acereal grain. A cereal grain includes any plant from the grass familythat yields an edible grain (seed). The most popular grains are barley,corn, millet, oats, quinoa, rice, rye, sorghum, triticale, wheat andwild rice. Examples of a cereal food product include, but are notlimited to: whole grain, crushed grain, grits, flour, bran, germ,breakfast cereals, extruded foods, pastas, and the like.

A baked goods product comprises any of the cereal food productsmentioned above and has been baked or processed in a manner comparableto baking (i.e., to dry or harden by subjecting to heat). Examples of abaked good product include, but are not limited to: bread, cakes,doughnuts, bars, pastas, bread crumbs, baked snacks, mini-biscuits,mini-crackers, mini-cookies, and mini-pretzels. As was mentioned above,oils of the invention can be used as an ingredient.

A snack food product comprises any of the above or below described foodproducts.

A fried food product comprises any of the above or below described foodproducts that has been fried.

A health food product is any food product that imparts a health benefit.Many oilseed-derived food products may be considered as health foods.

A beverage can be in a liquid or in a dry powdered form.

For example, there can be mentioned non-carbonated drinks such as fruitjuices, fresh, frozen, canned or concentrate; flavored or plain milkdrinks, etc. Adult and infant nutritional formulas are well known in theart and commercially available (e.g., Similac®, Ensure®, Jevity®, andAlimentum® from Ross Products Division, Abbott Laboratories).

Infant formulas are liquids or reconstituted powders fed to infants andyoung children. “Infant formula” is defined herein as an enteralnutritional product which can be substituted for human breast milk infeeding infants and typically is composed of a desired percentage of fatmixed with desired percentages of carbohydrates and proteins in anaqueous solution (e.g., see U.S. Pat. No. 4,670,285). Based on theworldwide composition studies, as well as levels specified by expertgroups, average human breast milk typically contains about 0.20% to0.40% of total fatty acids (assuming about 50% of calories from fat);and, generally the ratio of DHA to ARA would range from about 1:1 to 1:2(see, e.g., formulations of Enfamil LIPIL™ (Mead Johnson & Company) andSimilac Advance™ (Ross Products Division, Abbott Laboratories)). Infantformulas have a special role to play in the diets of infants becausethey are often the only source of nutrients for infants; and, althoughbreast-feeding is still the best nourishment for infants, infant formulais a close enough second that babies not only survive but thrive.

A dairy product is a product derived from milk. A milk analog ornondairy product is derived from a source other than milk, for example,soymilk as was discussed above. These products include, but are notlimited to: whole milk, skim milk, fermented milk products such asyogurt or sour milk, cream, butter, condensed milk, dehydrated milk,coffee whitener, coffee creamer, ice cream, cheese, etc.

Additional food products into which the PUFA-containing oils of theinvention could be included are, for example, chewing gums, confectionsand frostings, gelatins and puddings, hard and soft candies, jams andjellies, white granulated sugar, sugar substitutes, sweet sauces,toppings and syrups, and dry-blended powder mixes.

A health food product is any food product that imparts a health benefitand includes functional foods, medical foods, medical nutritionals anddietary supplements. Additionally, the plant/seed oils, altered seedsand microbial oils of the invention may be used in standardpharmaceutical compositions (e.g., the long-chain PUFA containing oilscould readily be incorporated into the any of the above mentioned foodproducts, to thereby produce a functional or medical food). Moreconcentrated formulations comprising PUFAs include capsules, powders,tablets, softgels, gelcaps, liquid concentrates and emulsions which canbe used as a dietary supplement in humans or animals other than humans.

Animal feeds are generically defined herein as products intended for useas feed or for mixing in feed for animals other than humans. Theplant/seed oils, altered seeds and microbial oils of the invention canbe used as an ingredient in various animal feeds.

More specifically, although not limited therein, it is expected that theoils of the invention can be used within pet food products, ruminant andpoultry food products and aquacultural food products. Pet food productsare those products intended to be fed to a pet (e.g., dog, cat, bird,reptile, and rodent). These products can include the cereal and healthfood products above, as well as meat and meat byproducts, soy proteinproducts, grass and hay products (e.g., alfalfa, timothy, oat or bromegrass, vegetables). Ruminant and poultry food products are those whereinthe product is intended to be fed to an animal (e.g., turkeys, chickens,cattle, and swine). As with the pet foods above, these products caninclude cereal and health food products, soy protein products, meat andmeat byproducts, and grass and hay products as listed above.Aquacultural food products (or “aquafeeds”) are those products intendedto be used in aquafarming, i.e., which concerns the propagation,cultivation, or farming of aquatic organisms and/or animals in fresh ormarine waters.

EXAMPLES

The present invention is further defined in the following Examples, inwhich parts and percentages are by weight and degrees are Celsius,unless otherwise stated. It should be understood that these Examples,while indicating preferred embodiments of the invention, are given byway of illustration only. From the above discussion and these Examples,one skilled in the art can ascertain the essential characteristics ofthis invention, and without departing from the spirit and scope thereof,can make various changes and modifications of the invention to adapt itto various usages and conditions. Thus, various modifications of theinvention in addition to those shown and described herein will beapparent to those skilled in the art from the foregoing description.Such modifications are also intended to fall within the scope of theappended claims.

The meaning of abbreviations is as follows: “sec” means second(s), “min”means minute(s), “h” means hour(s), “d” means day(s), “μL” meansmicroliter(s), “mL” means milliliter(s), “L” means liter(s), “μM” meansmicromolar, “mM” means millimolar, “M” means molar, “mmol” meansmillimole(s), “μmole” mean micromole(s), “g” means gram(s), “μg” meansmicrogram(s), “ng” means nanogram(s), “U” means unit(s), “bp” means basepair(s) and “kB” means kilobase(s).

General Methods: Nomenclature for Expression Cassettes:

The structure of an expression cassette will be represented by a simplenotation system of “X::Y::Z”, wherein X describes the promoter fragment,Y describes the gene coding region fragment, and Z describes theterminator fragment, which are all operably linked to one another.

Transformation and Cultivation of Yarrowia lipolytica:

Yarrowia lipolytica strains with ATCC Accession Nos. #20362, #76982 and#90812 were purchased from the American Type Culture Collection(Rockville, Md.). Yarrowia lipolytica strains were typically grown at28-30° C. in several media, according to the recipes shown below. Agarplates were prepared as required by addition of 20 g/L agar to eachliquid media, according to standard methodology.

YPD Agar Medium (Per Liter):

10 g of yeast extract [Difco], 20 g of Bacto peptone [Difco]; and 20 gof glucose.

Basic Minimal Media (MM) (Per Liter):

20 g glucose; 1.7 g yeast nitrogen base without amino acids; 1.0 gproline; and pH 6.1 (not adjusted).

Minimal Media+Uracil (MM+uracil or MMU) (Per Liter):

Prepare MM media as above and add 0.1 g uracil and 0.1 g uridine.

Minimal Media+Uracil+Sulfonylurea (MMU+SU) (Per Liter):

Prepare MMU media as above and add 280 mg sulfonylurea.

Minimal Media+Leucine (MM+Leucine or MMLeu) (Per Liter):

Prepare MM media as above and add 0.1 g leucine.

Minimal Media+Leucine+Uracil (MMLeuUra) (Per Liter):

Prepare MM media as above and add 0.1 g leucine, 0.1 g uracil and 0.1 guridine.

Minimal Media+Leucine+Lysine (MMLeuLys) (Per Liter):

Prepare MM media as above and add 0.1 g lysine, 0.1 g leucine.

Minimal Media+5-Fluoroorotic Acid (MM+5-FOA) (Per Liter):

20 g glucose, 6.7 g Yeast Nitrogen base, 75 mg uracil, 75 mg uridine andappropriate amount of FOA (Zymo Research Corp., Orange, Calif.), basedon FOA activity testing against a range of concentrations from 100 mg/Lto 1000 mg/L (since variation occurs within each batch received from thesupplier).

High Glucose Media (HGM) (Per Liter):

80 glucose, 2.58 g KH₂PO₄ and 5.36 g K₂HPO₄, pH 7.5 (do not need toadjust).

Transformation of Yarrowia lipolytica was performed according to themethod of Chen, D. C. et al. (Appl. Microbiol. Biotechnol. 48(2):232-235(1997)), unless otherwise noted. Briefly, Yarrowia was streaked onto aYPD plate and grown at 30° C. for approximately 18 h. Several largeloopfuls of cells were scraped from the plate and resuspended in 1 mL oftransformation buffer, comprising: 2.25 mL of 50% PEG, average MW 3350;0.125 mL of 2 M lithium acetate, pH 6.0; 0.125 mL of 2 M DTT; and(optionally) 50 μg sheared salmon sperm DNA. Then, approximately 500 ngof linearized plasmid DNA were incubated in 100 μL of resuspended cellsand maintained at 39° C. for 1 hr with vortex mixing at 15 minintervals. The cells were plated onto selection media plates, which weremaintained at 30° C. for 2 to 3 days.

Fatty Acid Analysis of Yarrowia lipolytica:

For fatty acid analysis, cells were collected by centrifugation andlipids were extracted as described in Bligh, E. G. & Dyer, W. J. (Can.J. Biochem. Physiol. 37:911-917 (1959)). Fatty acid methyl esters wereprepared by transesterification of the lipid extract with sodiummethoxide (Roughan, G. and Nishida I., Arch Biochem Biophys.276(1):38-46 (1990)) and subsequently analyzed with a Hewlett-Packard6890 GC fitted with a 30 m×0.25 mm (i.d.) HP-INNOWAX (Hewlett-Packard)column. The oven temperature was from 170° C. (25 min hold) to 185° C.at 3.5° C./min.

For direct base transesterification, Yarrowia culture (3 mL) washarvested, washed once in distilled water, and dried under vacuum in aSpeed-Vac for 5-10 min. Sodium methoxide (100 μL of 1%) was added to thesample, which was then vortexed and rocked for 20 min. After adding 3drops of 1 M NaCl and 400 μL hexane, the sample was vortexed and spun.The upper layer was removed and analyzed by GC as described above.

Construction Of Yarrowia lipolytica Strain Y4305U3:

Y. lipolytica strain Y4305U3 was used as the host in Examples 52, 53 and54, infra. The following description is a summary of the construction ofstrain Y4305U3, derived from Yarrowia lipolytica ATCC #20362. StrainY4305U3 is capable of producing about 53.2% EPA relative to the totallipids via expression of a delta-9 elongase/delta-8 desaturase pathway(FIG. 44).

The development of strain Y4305U3 required the construction of strainY2224 (a FOA resistant mutant from an autonomous mutation of the Ura3gene of wildtype Yarrowia strain ATCC #20362), strain Y4001 (producing17% EDA with a Leu− phenotype), strain Y4001U1 (producing 17% EDA with aLeu− and Ura− phenotype), strain Y4036 (producing 18% DGLA with a Leu−phenotype), strain Y4036U (producing 18% DGLA with a Leu− and Ura−phenotype), strain Y4070 (producing 12% ARA with a Ura− phenotype),strain Y4086 (producing 14% EPA), strain Y4086U1 (Ura3−), strain Y4128(producing 37% EPA), strain Y4128U3 (Ura−), strain Y4217 (producing 42%EPA), strain Y4217U2 (Ura−), strain Y4259 (producing 46.5% EPA) andstrain Y4259U2 (Ura−).

Generation Of Strain Y2224:

Strain Y2224 was isolated in the following manner: Yarrowia lipolyticaATCC #20362 cells from a YPD agar plate were streaked onto a MM plate(75 mg/L each of uracil and uridine, 6.7 g/L YNB with ammonia sulfate,without amino acids, and 20 g/L glucose) containing 250 mg/L 5-FOA (ZymoResearch). Plates were incubated at 28° C., and four of the resultingcolonies were patched separately onto MM plates containing 200 mg/mL5-FOA and MM plates lacking uracil and uridine. This was done to confirmuracil Ura3 auxotrophy.

Generation of Strain Y4001 to Produce about 17% EDA of Total Lipids:

Strain Y4001 was created via integration of construct pZKLeuN-29E3 (FIG.45A). This construct, comprising four chimeric genes (i.e., a delta-12desaturase, a C_(16/18) elongase, and two delta-9 elongases), wasintegrated into the Leu2 loci of strain Y2224 to thereby enableproduction of EDA.

Construct pZKLeuN-29E3 contained the components shown below in Table 8.

TABLE 8 Description of Plasmid pZKLeuN-29E3 (SEQ ID NO: 315) RE SitesAnd Nucleotides Within SEQ Description Of Fragment And Chimeric Gene IDNO: 315 Components BsiW I/Asc I 788 bp 3′ portion of Yarrowia Leu2 gene(GenBank (7797-7002) Accession No. AF260230) Sph I/Pac I 703 bp 5′portion of Yarrowia Leu2 gene (GenBank (4302-3591) Accession No.AF260230) Swa I/BsiW I GPD::FmD12::Pex20, comprising: (10533-7797) GPD:Yarrowia lipolytica GPD promoter (; U.S. Pat. No. 7,259,255); FmD12:Fusarium moniliforme delta-12 desaturase gene (SEQ ID NO: 316) (labeledas “F.D12” in Figure; PCT Publication No. WO 2005/047485); Pex20: Pex20terminator sequence from Yarrowia Pex20 gene (GenBank Accession No.AF054613) Bgl II/Swa I EXP1::EgD9eS::Lip1, comprising: (12559-10533)EXP1: Yarrowia lipolytica export protein (EXP1) promoter (labeled as“Exp pro” in Figure; U.S. patent application No. 11/265761); EgD9eS:codon-optimized delta-9 elongase (SEQ ID NO: 318), derived from Euglenagracilis (labeled as “EgD9E” in Figure; PCT Publication No. WO2007/061742); Lip1: Lip1 terminator sequence from Yarrowia Lip1 gene(GenBank Accession No. Z50020) Pme I/Cla I FBAINm::EgD9eS::Lip2,comprising: (12577-1) FBAINm: Yarrowia lipolytica FBAINm promoter (U.S.Pat. No. 7,202,356); EgD9eS: codon-optimized delta-9 elongase gene (SEQID NO: 318), derived from Euglena gracilis (labeled as “EgD9ES” inFigure; PCT Publication No. WO 2007/061742); Lip2: Lip2 terminatorsequence from Yarrowia Lip2 gene (GenBank Accession No. AJ012632) ClaI/EcoR I LoxP::Ura3::LoxP, comprising: (1-1736) LoxP sequence (SEQ IDNO: 320); Yarrowia Ura3 gene (GenBank Accession No. AJ306421); LoxPsequence (SEQ ID NO: 320) EcoR I/Pac I YAT1::ME3S::Pex16, comprising:(1736-3591) YAT1: Yarrowia lipolytica YAT1 promoter (labeled as “YAT” inFigure; Patent Publication No. U.S. 2006/0094102-A1); ME3S:codon-optimized C_(16/18) elongase gene (SEQ ID NO: 321), derived fromM. alpina (PCT Publication No. WO 2007/046817); Pex16: Pex16 terminatorsequence of Yarrowia Pex 16 gene (GenBank Accession No. U75433)

Plasmid pZKLeuN-29E3 was digested with AscI/SphI and then used fortransformation of Y. lipolytica strain Y2224 (i.e., ATCC #20362 Ura3-)according to the General Methods. The transformed cells were plated ontoMMLeu media plates, and plates were maintained at 30° C. for 2 to 3days. The colonies were picked and streaked onto MM and MMLeu selectionplates. The colonies that could grow on MMLeu plates but not on MMplates were selected as Leu− strains. Single colonies of Leu− strainswere used to inoculate liquid MMLeu, and the liquid cultures were shakenat 250 rpm/min for 2 days at 30° C. The cells were collected bycentrifugation, and lipids were extracted. Fatty acid methyl esters wereprepared by trans-esterification and subsequently analyzed with aHewlett-Packard 6890 GC.

GC analyses showed the presence of EDA in the transformants containingthe 4 chimeric genes of pZKLeuN-29E3, but not in the Yarrowia Y2224control strain. Most of the selected 36 Leu− strains produced about 12to 16.9% EDA of total lipids. Three strains, designated as strainsY4001, Y4002, and Y4003, produced about 17.4%, 17%, and 17.5% EDA oftotal lipids, respectively.

Single colonies of Y4001, Y4002, and Y4003 strains were used toinoculate liquid MMLeu, and the liquid cultures were shaken at 250rpm/min for 2 days at 30° C. The cells were collected by centrifugation,resuspended in HGM, and then shaken at 250 rpm/min for 5 days. The cellswere collected by centrifugation, and lipids were extracted. Fatty acidmethyl esters were prepared by trans-esterification and subsequentlyanalyzed with a Hewlett-Packard 6890 GC. GC analyses showed that theY4001, Y4002, and Y4003 strains produced about 24% EDA of total lipids.

Generation Of Strain Y4001U (Leu−, Ura−):

Strain Y4001U was created via temporary expression of the Crerecombinase enzyme in plasmid pY116 (FIG. 45B) within strain Y4001 toproduce a Leu− and Ura− phenotype. Construct pY116 contained thefollowing components:

TABLE 9 Description of Plasmid pY116 (SEQ ID NO: 323) RE Sites AndNucleotides Within SEQ Description Of Fragment And Chimeric Gene ID NO:323 Components 1328-448 ColE1 plasmid origin of replication 2258-1398Ampicillin-resistance gene (Amp^(R)) for selection in E. coli 3157-4461Yarrowia autonomous replication sequence (ARS18; GenBank Accession No.A17608) SwaI/PacI Yarrowia Leu2 gene (GenBank Accession No. 6667-4504AF260230) Swa I/Pme I GPAT::Cre::XPR2, comprising: (6667-218) GPAT:Yarrowia lipolytica GPAT promoter (U.S. Pat. No. 7,264,949); Cre:Enterobacteria phage P1 Cre gene for recombinase protein (GenBankAccession No. X03453); XPR2: ~100 bp of the 3′ region of the YarrowiaXpr gene (GenBank Accession No. M17741)

Plasmid pY116 was used for transformation of freshly grown Y4001 cellsaccording to the General Methods. The transformed cells were plated ontoMMLeuUra plates containing 280 μg/mL sulfonylurea (chlorimuron ethyl, E.I. duPont de Nemours & Co., Inc., Wilmington, Del.), and plates weremaintained at 30° C. for 3 to 4 days. Four colonies were picked and thenused to inoculate 3 mL liquid YPD. The liquid cultures were shaken at250 rpm/min for 1 day at 30° C. The cultures were diluted to 1:50,000with liquid MMLeuUra media, and 100 μL were plated onto new YPD plates.The plates were maintained at 30° C. for 2 days. Colonies were pickedand streaked onto MMLeu and MMLeuUra selection plates. The colonies thatcould grow on MMLeuUra plates but not on MMLeu plates were selected andanalyzed by GC to confirm the presence of C20:2 (EDA). Several strains,each having a Leu− and Ura− phenotype, produced about 17% EDA of totallipids and collectively, were designated as Y4001U. One of these strainswas designated as Y4001U1.

Generation Of Y4036 Strain to Produce About 18% DGLA Of Total Lipids:

Construct pKO2UF8289 (FIG. 46A; SEQ ID NO:324) was generated tointegrate four chimeric genes (comprising a delta-12 desaturase, onedelta-9 elongase, and two mutant delta-8 desaturases) into the delta-12loci of strain Y4001U1, to thereby enable production of DGLA. ConstructpKO2UF8289 contained the following components:

TABLE 10 Description of Plasmid pKO2UF8289 (SEQ ID NO: 324) RE Sites AndNucleotides Within SEQ Description Of Fragment And Chimeric Gene ID NO:324 Components AscI/BsiWI 5′ portion of Yarrowia delta-12 desaturasegene (SEQ (10337-9600) ID NO: 325) (labeled as “YLD12-N” in Figure; U.S.Pat. No. 7,214,491) EcoRI/SphI 3′ portion of Yarrowia delta-12desaturase gene (SEQ (13601-13045) ID NO: 325) (labeled as “YL12-C” inFigure; PCT Publication No. WO 2004/104167; U.S. Pat. No. 7,214,491)SwaI/BsiWI FBAINm::EgD8M::Pex20, comprising: (7088-9600) FBAINm:Yarrowia lipolytica FBAINm promoter (PCT Publication No. WO 2005/049805;U.S. Pat. No. 7,202,356); EgD8M: Synthetic mutant delta-8 desaturase(SEQ ID NO: 327) (labeled as “D8S-23” in Figure; U.S. patent applicationNo. 11/635258), derived from Euglena gracilis (“EgD8S”; U.S. Pat. No.7,256,033); Pex20: Pex20 terminator sequence from Yarrowia Pex20 gene(GenBank Accession No. AF054613) SwaI/PmeI YAT1::FmD12::OCT, comprising:(7088-4581) YAT1: Yarrowia lipolytica YAT1 promoter (labeled as “YAT” inFigure; Patent Publication No. US 2006/0094102-A1); FmD12: Fusariummoniliforme delta-12 desaturase gene (SEQ ID NO: 316) (labeled as“F.D12” in Figure; PCT Publication No. WO 2005/047485); OCT: OCTterminator sequence of Yarrowia OCT gene (GenBank Accession No. X69988)PmeI/PacI EXP1::EgD8M::Pex16, comprising: (4581-2124) EXP1: Yarrowialipolytica export protein (EXP1) promoter (PCT Publication No. WO2006/052870 and U.S. patent application No. 11/265761); EgD8M: Syntheticmutant delta-8 desaturase (SEQ ID NO: 327) (labeled as “D8-23” inFigure; U.S. patent application No. 11/635258), derived from Euglenagracilis (“EgD8S”; PCT Publication No. WO 2006/012326; U.S. Pat. No.7,256,033); Pex16: Pex16 terminator sequence from Yarrowia Pex16 gene(GenBank Accession No. U75433) PmeI/ClaI GPAT::EgD9e::Lip2, comprising:(2038-1) GPAT: Yarrowia lipolytica GPAT promoter (PCT Publication No. WO2006/031937; U.S. Pat. No. 7,264,949); EgD9e: Euglena gracilis delta-9elongase gene (SEQ ID NO: 329) (labeled as “EgD9E” in Figure; PCTPublication No. WO 2007/061742); Lip2: Lip2 terminator sequence fromYarrowia Lip2 gene (GenBank Accession No. AJ012632) ClaI/EcoRILoxP::Ura3::LoxP, comprising: (13601-1) LoxP sequence (SEQ ID NO: 320);Yarrowia Ura3 gene (GenBank Accession No. AJ306421); LoxP sequence (SEQID NO: 320)

The pKO2UF8289 plasmid was digested with AscI/SphI and then used fortransformation of strain Y4001U1 according to the General Methods. Thetransformed cells were plated onto MMLeu plates, and plates weremaintained at 30° C. for 2 to 3 days. The colonies were picked andstreaked onto MMLeu selection plates at 30° C. for 2 days. These cellswere then used to inoculate liquid MMLeu media, and liquid cultures wereshaken at 250 rpm/min for 2 days at 30° C. The cells were collected bycentrifugation, and lipids were extracted. Fatty acid methyl esters wereprepared by trans-esterification and subsequently analyzed with aHewlett-Packard 6890 GC.

GC analyses showed the presence of DGLA in the transformants containingthe 4 chimeric genes of pKO2UF8289, but not in the parent Y4001U1strain. Most of the selected 96 strains produced between 7% and 13% DGLAof total lipids. Six strains, designated as Y4034, Y4035, Y4036, Y4037,Y4038, and Y4039, produced about 15%, 13.8%, 18.2%, 13.1%, 15.6%, and13.9% DGLA of total lipids, respectively.

Generation Of Strain Y4036U (Leu−, Ura3−):

Construct pY116 (FIG. 45B; SEQ ID NO:323) was utilized to temporarilyexpress a Cre recombinase enzyme in strain Y4036. This released the LoxPsandwiched Ura3 gene from the genome.

Plasmid pY116 was used to transform strain Y4036 according to theGeneral Methods. Following transformation, the cells were plated ontoMMLeuUra plates, and plates were maintained at 30° C. for 2 to 3 days.The individual colonies grown on MMLeuUra plates were picked andstreaked into YPD liquid media. Liquid cultures were shaken at 250rpm/min for 1 day at 30° C. to cure the pY116 plasmid. Cells from thegrown cultures were streaked on MMLeuUra plates. After two days at 30°C., the individual colonies were re-streaked on MMLeuUra, MMU and MMLeuplates. Those colonies that could grow on MMLeuUra, but not on MMU orMMLeu plates, were selected. One strain with Leu− and Ura− phenotypeswas designated as Y4036U (Ura−, Leu−).

Generation of Y4069 and Y4070 Strains to Produce about 12% ARA of TotalLipids:

Construct pZKSL-555R (FIG. 46B; SEQ ID NO:331) was generated tointegrate three delta-5 desaturase genes into the Lys loci of strainY4036U, to thereby enable production of ARA. The pZKSL-555R plasmidcontained the following components:

TABLE 11 Description of Plasmid pZKSL-555R (SEQ ID NO: 331) RE Sites AndNucleotides Within SEQ Description Of Fragment And Chimeric Gene ID No:331 Components AscI/BsiWI 720 bp 5′ portion of Yarrowia Lys5 gene(GenBank (3321-2601) Accession No. M34929) PacI/SphI 687 bp 3′ portionof Yarrowia Lys5 gene (Gen Bank (6716-6029) Accession No. M34929)BglII/BsiWI EXP1::EgD5S::Pex20, comprising: (15-2601) EXP1: Yarrowialipolytica export protein (EXP1) promoter (labeled as “EXP” in Figure;U.S. patent application No. 11/265761); EgD5S: codon-optimized delta-5desaturase (SEQ ID NO: 332), derived from Euglena gracilis (PatentPublication US 2007-0292924-A1); Pex20: Pex20 terminator sequence fromYarrowia Pex20 gene (GenBank Accession No. AF054613) ClaI/PmeIYAT1::RD5S::OCT, comprising: (11243-1) YAT1: Yarrowia lipolytica YAT1promoter (labeled as “YAT” in Figure; Patent Publication US2006/0094102-A1); RD5S: codon-optimized delta-5 desaturase (SEQ ID NO:334), derived from Peridinium sp. CCMP626 (labeled as “RD5S(626)” inFigure; Patent Publication US 2007-0271632-A1); OCT: OCT terminatorsequence of Yarrowia OCT gene (GenBank Accession No. X69988) EcoRI/PacIFBAIN::EgD5::Aco, comprising: (9500-6716) FBAIN: Yarrowia lipolyticaFBAIN promoter (U.S. Pat. No. 7,202,356); EgD5: Euglena gracilis delta-5desaturase (SEQ ID NO: 336) (labeled as “EgD5WT” in Figure; PatentPublication US 2007-0292924-A1) with elimination of internal EcoRI,BglII, HindIII and NcoI restriction enzyme sites [mutations labeled as“M.EI”, “M.BII”, “M.H” and “M.N”, respectively]; Aco: Aco terminatorsequence from Yarrowia Aco gene (GenBank Accession No. AJ001300)EcoRI/ClaI Yarrowia Leu2 gene (GenBank Accession No. (9500-11243)M37309)

The pZKSL-555R plasmid was digested with AscI/SphI and then used fortransformation of strain Y4036U according to the General Methods. Thetransformed cells were plated onto MMLeuLys plates, and plates weremaintained at 30° C. for 2 to 3 days. Single colonies were thenre-streaked onto MMLeuLys plates, and the resulting colonies were usedto inoculate liquid MMLeuLys. Liquid cultures were then shaken at 250rpm/min for 2 days at 30° C. The cells were collected by centrifugation,and lipids were extracted. Fatty acid methyl esters were prepared bytrans-esterification and subsequently analyzed with a Hewlett-Packard6890 GC.

GC analyses showed the presence of ARA in the transformants containingthe 3 chimeric genes of pZKSL-555R, but not in the parent Y4036U strain.Most of the selected 96 strains produced ˜10% ARA of total lipids. Fourstrains, designated as Y4068, Y4069, Y4070, and Y4071, produced about11.7%, 11.8%, 11.9% and 11.7% ARA of total lipids, respectively. Furtheranalyses showed that the three chimeric genes of pZKSL-555R were notintegrated into the Lys5 site in the Y4068, Y4069, Y4070 and Y4071strains. All strains possessed a Lys+ phenotype.

The final genotype of strain Y4070, with respect to wildtype Yarrowialipolytica ATCC #20362, was Ura−, unknown 1-, unknown 3-, Leu+, Lys+,GPD::FmD12::Pex20, YAT1::FmD12::OCT, YAT1::ME3S::Pex16,GPAT::EgD9e::Lip2, EXP1::EgD9eS::Lip1, FBAINm::EgD9eS::Lip2,FBAINm::EgD8M::Pex20, EXP1::EgD8M::Pex16, FBAIN::EgD5::Aco,EXP1::EgD5S::Pex20, YAT1::RD5S::OCT.

Generation of Y4086 Strain to Produce about 14% EPA of Total Lipids:

Construct pZP3-Pa777U (FIG. 47A; SEQ ID NO:338) was generated tointegrate three delta-17 desaturase genes into the Pox3 loci (GenBankAccession No. AJ001301) of strain Y4070, to thereby enable production ofEPA. The pZP3-Pa777U plasmid contained the following components:

TABLE 12 Description of Plasmid pZP3-Pa777U (SEQ ID NO: 338) RE SitesAnd Nucleotides Within SEQ Description Of Fragment And Chimeric Gene IDNO: 338 Components AscI/BsiWI 770 bp 5′ portion of Yarrowia Pox3 gene(GenBank (3527-4297) Accession No. AJ001301) PacI/SphI 827 bp 3′ portionof Yarrowia Pox3 gene (GenBank (1-827) Accession No. AJ001301)ClaI/SwaWI YAT1::PaD17S::Lip1, comprising: (6624-4457) YAT1: Yarrowialipolytica YAT1 promoter (labeled as “YAT” in Figure; Patent PublicationUS 2006/0094102-A1); PaD17S: codon-optimized delta-17 desaturase (SEQ IDNO: 339), derived from Pythium aphanidermatum (U.S. patent applicationNo. 11/779915); Lip1: Lip1 terminator sequence from Yarrowia Lip1 gene(GenBank Accession No. Z50020) EcoRI/PmeI EXP1::PaD17::Pex16,comprising: (8359-10611) EXP1: Yarrowia lipolytica export protein (EXP1)promoter (labeled as “Exp” in Figure; U.S. patent application No.11/265761); PaD17: Pythium aphanidermatum delta-17 desaturase gene (SEQID NO: 341) (labeled as “PaD17WT” in Figure; U.S. patent application No.11/779915); Pex16: Pex16 terminator sequence from Yarrowia Pex16 gene(GenBank Accession No. U75433) PmeI/PacI FBAINm::PaD17::Aco, comprising:(10611-1) FBAINm: Yarrowia lipolytica FBAINm promoter (WO 2005/049805;U.S. Pat. No. 7,202,356); PaD17: Pythium aphanidermatum delta-17desaturase gene (SEQ ID NO: 341) (labeled as “PaD17WT” in Figure; U.S.patent application No. 11/779915); Aco: Aco terminator sequence fromYarrowia Aco gene (GenBank Accession No. AJ001300) ClaI/EcoRILoxP::Ura3::LoxP, comprising: (6624-8359) LoxP sequence (SEQ ID NO:320); Yarrowia Ura3 gene (GenBank Accession No. AJ306421); LoxP sequence(SEQ ID NO: 320)

The pZP3-Pa777U plasmid was digested with AscI/SphI and then used fortransformation of strain Y4070 according to the General Methods. Thetransformed cells were plated onto MM plates, and plates were maintainedat 30° C. for 2 to 3 days. Single colonies were then re-streaked onto MMplates, and the resulting colonies were used to inoculate liquidMMLeuLys. Liquid cultures were shaken at 250 rpm/min for 2 days at 30°C. The cells were collected by centrifugation, and lipids wereextracted. Fatty acid methyl esters were prepared bytrans-esterification and subsequently analyzed with a Hewlett-Packard6890 GC.

GC analyses showed the presence of EPA in the transformants containingthe 3 chimeric genes of pZP3-Pa777U, but not in the parent Y4070 strain.Most of the selected 96 strains produced 10-13% EPA of total lipids. Twostrains, designated as Y4085 and Y4086, produced about 14.2% and 13.8%EPA of total lipids, respectively.

The final genotype of strain Y4086, with respect to wildtype Yarrowialipolytica ATCC #20362, was Ura3+, Leu+, Lys+, unknown 1-, unknown 2-,YALI0F24167g-, GPD::FmD12::Pex20, YAT1::FmD12::OCT, YAT1::ME3S::Pex16,GPAT::EgD9e::Lip2, EXP1::EgD9eS::Lip1, FBAINm::EgD9eS::Lip2,FBAINm::EgD8M::Pex20, EXP1::EgD8M::Pex16, FBAIN::EgD5::Aco,EXP1::EgD5S::Pex20, YAT1::RD5S::OCT, YAT1::PaD17S::Lip1,EXP1::PaD17::Pex16, FBAINm::PaD17::Aco.

Generation of Strain Y4086U1 (Ura3-):

Strain Y4086U1 was created via temporary expression of the Crerecombinase enzyme in construct pY117 (FIG. 47B; SEQ ID NO:343) withinstrain Y4086 to produce a Ura− phenotype. This released the LoxPsandwiched Ura3 gene from the genome. The mutated Yarrowia AHAS enzymein plasmid pY117 conferred SU^(R), which was used as a positivescreening marker.

Plasmid pY117 was derived from plasmid pY116 (described supra, and inU.S. patent application Ser. No. 11/635,258) by inserting the mutantAHAS gene flanked by PacI-SwaI sites into PacI-SwaI digested pY116,thereby replacing the LEU selectable marker with the sulfonylureamarker. Construct pY117 thereby contained the following components:

TABLE 13 Description of Plasmid pY117 (SEQ ID NO: 343) RE Sites AndNucleotides Within SEQ Description Of Fragment And Chimeric Gene ID NO:343 Components 1328-448 ColE1 plasmid origin of replication 2258-1398Ampicillin-resistance gene (Amp^(R)) for selection in E. coli 2438-2838E. coli f1 origin of replication 3157-4461 Yarrowia autonomousreplication sequence (ARS18; GenBank Accession No. A17608) PacI/SwaIYarrowia lipolytica AHAS gene (GenBank Accession 4504-7498 No.XP_501277) comprising a W497L mutation (SEQ ID NO: 344; PCT PublicationNo. WO 2006/052870) SwaI/PmeI GPAT::Cre::XPR, comprising: 7498-218 GPAT:Yarrowia lipolytica GPAT promoter (PCT Publication No. WO 2006/031937;U.S. Pat. No. 7,264,949); Cre: Enterobacteria phage P1 Cre gene forrecombinase protein (GenBank Accession No. X03453) except for singlebase change (T4G) resulting in a single amino acid change (S2A) tocreate a NcoI site for cloning convenience; XPR: ~100 bp of the 3′region of the Yarrowia Xpr gene (GenBank Accession No. M17741)

Plasmid pY117 was used to transform strain Y4086 according to theGeneral Methods. Following transformation, the cells were plated ontoMMU+SU (280 μg/mL sulfonylurea; also known as chlorimuron ethyl, E. I.duPont de Nemours & Co., Inc., Wilmington, Del.) plates, and plates weremaintained at 30° C. for 2 to 3 days. The individual SU^(R) coloniesgrown on MMU+SU plates were picked and streaked into YPD liquid media,and liquid cultures were shaken at 250 rpm/min for 1 day at 30° C. tocure the pY117 plasmid. Cells from the grown cultures were streaked ontoMMU plates. After two days at 30° C., the individual colonies werere-streaked onto MM and MMU plates. Those colonies that could grow onMMU, but not on MM plates were selected. Two of these strains with Ura−phenotypes were designated as Y4086U1 and Y4086U2 (Ura−).

Generation of Y4128 Strain to Produce about 37% EPA of Total Lipids:

Construct pZP2-2988 (FIG. 48A; SEQ ID NO:345) was generated to integrateone delta-12 desaturase gene, two delta-8 desaturase genes, and onedelta-9 elongase gene into the Pox2 loci (GenBank Accession No.AJ001300) of strain Y4086U1, to thereby enable higher level productionof EPA. The pZP2-2988 plasmid contained the following components:

TABLE 14 Description of Plasmid pZP2-2988 (SEQ ID NO: 345) RE Sites AndNucleotides Within SEQ Description Of Fragment And Chimeric Gene ID NO:345 Components AscI/BsiWI 803 bp 5′ portion of Yarrowia Pox2 gene(GenBank (3083-2273) Accession No. AJ001300) PacI/SphI 649 bp 3′ portionof Yarrowia Pox2 gene (GenBank (6446-5791) Accession No. AJ001300)PmeI/BsiWI FBA::EgD9eS::Pex20, comprising: (347-2273) FBA: Yarrowialipolytica FBA promoter (PCT Publication No. WO 2005/049805; U.S. Pat.No. 7,202,356); EgD9eS: codon-optimized delta-9 elongase (SEQ ID NO:318), derived from Euglena gracilis (PCT Publication No. WO2007/061742); Pex20: Pex20 terminator sequence from Yarrowia Pex20 gene(GenBank Accession No. AF054613) ClaI/PmeI GPM/FBAIN::FmD12S::OCT,comprising: (13318-347) GPM/FBAIN: chimeric Yarrowia lipolyticaGPM/FBAIN promoter (separately labeled as “GPM” and “FBA intron” inFigure) (PCT Publication No. WO 2005/049805; U.S. Pat. No. 7,202,356);FmD12S: codon-optimized delta-12 desaturase (SEQ ID NO: 346), derivedfrom Fusarium moniliforme (labeled as “F.D12S” in Figure; PCTPublication No. WO 2005/047485); OCT: OCT terminator sequence ofYarrowia OCT gene (GenBank Accession No. X69988) ClaI/EcoRILoxP::Ura3::LoxP, comprising: (13318-11581) LoxP sequence (SEQ ID NO:320); Yarrowia Ura3 gene (GenBank Accession No. AJ306421); LoxP sequence(SEQ ID NO: 320) EcoRII/SwaI GPDIN::EgD8M::Lip1, comprising:(11581-8884) GPDIN: Yarrowia lipolytica GPDIN promoter (PatentPublication US 2006/0019297-A1); EgD8M: Synthetic mutant delta-8desaturase (SEQ ID NO: 327; U.S. patent application No. 11/635258),derived from Euglena gracilis (“EgD8S”; U.S. Pat. No. 7,256,033); Lip1:Lip1 terminator sequence from Yarrowia Lip1 gene (GenBank Accession No.Z50020) SwaI/PacI YAT1::EgD8M::ACO, comprising: (8884-6446) YAT1:Yarrowia lipolytica YAT1 promoter (labeled as “YAT” in Figure; PatentPublication US 2006/0094102-A1); EgD8M: Synthetic mutant delta-8desaturase (SEQ ID NO: 327; U.S. patent application No. 11/635258),derived from Euglena gracilis (“EgD8S”; U.S. Pat. No. 7,256,033); Aco:Aco terminator sequence from Yarrowia Aco gene (GenBank Accession No.AJ001300)

The pZP2-2988 plasmid was digested with AscI/SphI and then used fortransformation of strain Y4086U1 according to the General Methods. Thetransformed cells were plated onto MM plates, and plates were maintainedat 30° C. for 2 to 3 days. Single colonies were re-streaked onto MMplates, and the resulting colonies were used to inoculate liquidMMLeuLys. Liquid cultures were shaken at 250 rpm/min for 2 days at 30°C. The cells were collected by centrifugation, resuspended in HGM, andthen shaken at 250 rpm/min for 5 days. The cells were collected bycentrifugation, and lipids were extracted. Fatty acid methyl esters wereprepared by trans-esterification and subsequently analyzed with aHewlett-Packard 6890 GC.

GC analyses showed that most of the selected 96 strains produced12-15.6% EPA of total lipids. Two strains, designated as Y4128 andY4129, produced about 37.6% and 16.3% EPA of total lipids, respectively.

The final genotype of strain Y4128, with respect to wildtype Yarrowialipolytica ATCC #20362, was: YALI0F24167g-, Pex10-, unknown 1-, unknown2-, GPD::FmD12::Pex20, YAT1::FmD12::OCT, GPM/FBAIN::FmD12S::OCT,YAT1::ME3S::Pex16, GPAT::EgD9e::Lip2, EXP1::EgD9eS::Lip1,FBAINm::EgD9eS::Lip2, FBA::EgD9eS::Pex20, FBAINm::EgD8M::Pex20,EXP1::EgD8M::Pex16, GPDIN::EgD8M::Lip1, YAT1::EgD8M::Aco,FBAIN::EgD5::Aco, EXP1::EgD5S::Pex20, YAT1::RD5S::OCT,YAT1::PaD17S::Lip1, EXP1::PaD17::Pex16, FBAINm::PaD17::Aco. Yarrowialipolytica strain Y4128 was deposited with the American Type CultureCollection on Aug. 23, 2007 and bears the designation ATCC PTA-8614.

Generation of Y4128U Strains:

In order to disrupt the Ura3 gene in strain Y4128, construct pZKUE3S(FIG. 48B; SEQ ID NO:351) was created to integrate a EXP1::ME3S::Pex20chimeric gene into the Ura3 gene of strain Y4128. Plasmid pZKUE3Scontained the following components:

TABLE 15 Description of Plasmid pZKUE3S (SEQ ID NO: 351) RE Sites AndNucleotides Within SEQ Description Of Fragment And Chimeric Gene ID NO:351 Components BsiWI/PacI 721 bp 5′ portion of Yarrowia Ura3 gene(GenBank (318-1038) Accession No. AJ306421) SphI/PmeI 729 bp 3′ portionof Yarrowia Ura3 gene (GenBank (3915-4594) Accession No. AJ306421)EcoRI/BsiWI EXP1::ME3S::Pex20, comprising: (4628-318) EXP1: Yarrowialipolytica export protein (EXP1) promoter (labeled as “Exp” in Figure;U.S. patent application No. 11/265761); ME3S: codon-optimized C_(16/18)elongase gene (SEQ ID NO: 321), derived from Mortierella alpina (PCTPublication No. WO 2007/046817); Pex20: Pex20 terminator sequence ofYarrowia Pex20 gene (GenBank Accession No. AF054613) 2149-1269 ColE1plasmid origin of replication 3079-2219 ampicillin-resistance gene(Amp^(R)) for selection in E. coli 3659-3259 f1 origin

Plasmid pZKUE3S was digested with SphI/PacI and then used to transformstrain Y4128 according to the General Methods. Following transformation,cells were plated onto MM+5-FOA selection plates, and plates weremaintained at 30′C for 2 to 3 days.

A total of 24 transformants grown on MM+5-FOA selection plates werepicked and re-streaked onto fresh MM+5-FOA plates. The cells werestripped from the plates, and lipids were extracted. Fatty acid methylesters were prepared by trans-esterification and subsequently analyzedwith a Hewlett-Packard 6890 GC.

GC analyses showed the presence of between 10-15% EPA in all of thetransformants with pZKUE3S from plates. The strains designated asY4128U1, Y4128U2, Y4128U3, Y4128U4, Y4128U5, and Y4128U6 produced 12.9%,14.4%, 15.2%, 15.4%, 14%, and 10.9% EPA, respectively (collectively,Y4128U).

The discrepancy in the % EPA quantified in Y4128 (37.6%) versus Y4128U(average 13.8%) is based on differing growth conditions. Specifically,the former culture was analyzed following two days of growth in liquidculture, while the latter culture was analyzed after growth on an agarplate. The Applicants have observed a 2-3 fold increase in % EPA, whencomparing results from agar plates to those in liquid culture. Thus,although results are not directly comparable, both Y4128 and Y4128Ustrains demonstrate high production of EPA.

Generation of Y4217 Strain to Produce about 42% EPA of Total Lipids:

Construct pZKL2-5U89GC (FIG. 49A; SEQ ID NO:348) was generated tointegrate one delta-9 elongase gene, one delta-8 desaturase gene, onedelta-5 desaturase gene, and one Yarrowia lipolytica diacylglycerolcholinephosphotransferase gene (CPT1) into the Lip2 loci (GenBankAccession No. AJ012632) of strain Y4128U3 to thereby enable higher levelproduction of EPA. The pZKL2-5U89GC plasmid contained the followingcomponents:

TABLE 16 Description of Plasmid pZKL2-5U89GC (SEQ ID NO: 348) RE SitesAnd Nucleotides Within SEQ Description Of Fragment And Chimeric Gene IDNO: 348 Components AscI/BsiWI 722 bp 5′ portion of Yarrowia Lip2 gene(labeled as (730-1) “Lip2.5N” in Figure; GenBank Accession No. AJ012632)PacI/SphI 697 bp 3′ portion of Yarrowia Lip2 gene (labeled as(4141-3438) “Lip2.3N” in Figure; GenBank Accession No. AJ012632)SwaI/BsiWI YAT1::YICPT1::Aco, comprising: (13382-1) YAT1: Yarrowialipolytica YAT1 promoter (labeled as “YAT” in Figure; Patent PublicationUS 2006/0094102-A1); YICPT1: Yarrowia lipolytica diacylglycerolcholinephosphotransferase gene (SEQ ID NO: 349) (labeled as “CPT1” inFigure; PCT Publication No. WO 2006/052870); Aco: Aco terminatorsequence from Yarrowia Aco gene (GenBank Accession No. AJ001300)PmeI/SwaI FBAIN::EgD8M::Lip1 comprising: (10745-13382) FBAIN: Yarrowialipolytica FBAIN promoter (U.S. Pat. No. 7,202,356); EgD8M: Syntheticmutant delta-8 desaturase (SEQ ID NO: 327) (labeled as “D8S-23” inFigure; U.S. patent application No. 11/635258), derived from Euglenagracilis (“EgD8S”; U.S. Pat. No. 7,256,033); Lip1: Lip1 terminatorsequence from Yarrowia Lip1 gene (GenBank Accession No. Z50020)PmeI/ClaI GPD::EgD9eS::Lip2, comprising: (10745-8650) GPD: Yarrowialipolytica GPD promoter (labeled as “GPD Pro” in Figure; U.S. Pat. No.7,259,255); EgD9eS: codon-optimized delta-9 elongase gene (SEQ ID NO:318), derived from Euglena gracilis (labeled as “EgD9ES” in Figure; PCTPublication No. WO 2007/061742); Lip2: Lip2 terminator sequence fromYarrowia Lip2 gene (GenBank Accession No. AJ012632) ClaI/EcoRI YarrowiaUra3 gene (GenBank Accession No. (8650-6581) AJ306421) EcoRI/PacIYAT1::EgDD5S::ACO, comprising: (6581-4141) YAT1: Yarrowia lipolyticaYAT1 promoter (labeled as “YAT” in Figure; Patent Publication US2006/0094102-A1); EgD5S: codon-optimized delta-5 desaturase (SEQ ID NO:332), derived from Euglena gracilis (Patent Publication US2007-0292924-A1); Aco: Aco terminator sequence from Yarrowia Aco gene(GenBank Accession No. AJ001300)

The pZKL2-5U89GC plasmid was digested with AscI/SphI and then used fortransformation of strain Y4128U3 according to the General Methods. Thetransformed cells were plated onto MM plates, and plates were maintainedat 30° C. for 3 to 4 days. Single colonies were re-streaked onto MMplates, and the resulting colonies were then used to inoculate liquidMM. Liquid cultures were shaken at 250 rpm/min for 2 days at 30° C. Thecells were collected by centrifugation, resuspended in HGM, and thenshaken at 250 rpm/min for 5 days. The cells were collected bycentrifugation, and lipids were extracted. Fatty acid methyl esters wereprepared by trans-esterification and subsequently analyzed with aHewlett-Packard 6890 GC.

GC analyses showed that most of the selected 96 strains produced32-39.9% EPA of total lipids. Six strains, designated as Y4215, Y4216,Y4217, Y4218, Y4219 and Y4220, produced about 41.1%, 41.8%, 41.7%,41.1%, 41% and 41.1% EPA of total lipids, respectively. The finalgenotype of each strain, with respect to wild type Yarrowia lipolyticaATCC #20362, was: YALI0C18711g-, Pex10-, YALI0F24167g-, unknown 1-,unknown 3-, GPD::FmD12::Pex20, YAT1::FmD12::OCT, GPM/FBAIN::FmD12S::OCT,YAT1::ME3S::Pex16, EXP1::ME3S::Pex20, GPAT::EgD9e::Lip2,EXP1::EgD9eS::Lip1, FBAINm::EgD9eS::Lip2, FBA::EgD9eS::Pex20,GPD::EgD9eS::Lip2, FBAINm::EgD8M::Pex20, FBAIN::EgD8M::Lip1,EXP1::EgD8M::Pex16, GPDIN::EgD8M::Lip1, YAT1::EgD8M::Aco,FBAIN::EgD5::Aco, EXP1::EgD5S::Pex20, YAT1::EgD5S::Aco, YAT1::RD5S::OCT,YAT1::PaD17S::Lip1, EXP1::PaD17::Pex16, FBAINm::PaD17::Aco,YAT1::YICPT1::ACO.

Generation of Strain Y4217U2 (Ura3-):

In order to disrupt the Ura3 gene in strain Y4217, construct pZKUE3S(FIG. 48B; SEQ ID NO:351) was used to integrate a chimericEXP1::ME3S::Pex20 gene into the Ura3 gene of strain Y4217. Followingtransformation, cells were plated onto MM+5-FOA selection plates, andplates were maintained at 30° C. for 3 to 4 days.

A total of 6 transformants grown on MM+5-FOA plates were picked andre-streaked onto MM plates and MM+5-FOA plates. All 6 strains had aUra−phenotype (i.e., cells could grow on MM+5-FOA plates, but not on MMplates). The cells were scraped from the MM+5-FOA plates, and lipidswere extracted. Fatty acid methyl esters were prepared bytrans-esterification and subsequently analyzed with a Hewlett-Packard6890 GC.

GC analyses showed the presence of 18.7% to 28.6% EPA in all of thetransformants with pZKUE3S grown on MM+5-FOA plates. Two strains,designated as strains Y4217U1 and Y4217U2, produced 22.5% and 28.6% EPA,respectively.

Generation of Y4259 Strain to Produce about 46.5% EPA of Total Lipids:

Construct pZKL1-2SP98C (FIG. 49B; SEQ ID NO:352) was generated tointegrate one delta-9 elongase gene, one delta-8 desaturase gene, onedelta-12 desaturase gene, and one Yarrowia lipolytica diacylglycerolcholinephosphotransferase gene (CPT1) into the Lip1 loci (GenBankAccession No. Z50020) of strain Y4217U2, to thereby enable higher levelproduction of EPA. The pZKL1-2SP98C plasmid contained the followingcomponents:

TABLE 17 Description of Plasmid pZKL1-2SP98C (SEQ ID NO: 352) RE SitesAnd Nucleotides Within SEQ Description Of Fragment And Chimeric Gene IDNO: 352 Components AscI/BsiWI 809 bp 5′ portion of Yarrowia Lip1 gene(labeled as (3474-2658) “Lip1-5′N” in Figure; GenBank Accession No.Z50020) PacI/SphI 763 bp 3′ portion of Yarrowia Lip1 gene (labeled as(6951-6182) “Lip1.3N” in Figure; GenBank Accession No. Z50020)SwaI/BsiWI GPD::YICPT1::Aco, comprising: (1-2658) GPD: Yarrowialipolytica GPD promoter (U.S. Pat. No. 7,259,255); YICPT1: Yarrowialipolytica diacylglycerol cholinephosphotransferase gene (SEQ ID NO:349) (labeled as “CPT1” in Figure; PCT Publication No. WO 2006/052870);Aco: Aco terminator sequence from Yarrowia Aco gene (GenBank AccessionNo. AJ001300) PmeI/SwaI FBAIN::EgD8M::Lip1 comprising: (13241-1) FBAIN:Yarrowia lipolytica FBAIN promoter (U.S. Pat. No. 7,202,356); EgD8M:Synthetic mutant delta-8 desaturase (SEQ ID NO: 327; U.S. patentapplication No. 11/635258), derived from Euglena gracilis (“EgD8S”; U.S.Pat. No. 7,256,033); Lip1: Lip1 terminator sequence from Yarrowia Lip1gene (GenBank Accession No. Z50020) PmeI/ClaI YAT1::EgD9eS::Lip2,comprising: (13241-11385) YAT1: Yarrowia lipolytica YAT1 promoter(labeled as “YAT” in Figure; Patent Publication US 2006/0094102-A1);EgD9eS: codon-optimized delta-9 elongase gene (SEQ ID NO: 318), derivedfrom Euglena gracilis (labeled as “EgD9ES” in Figure; PCT PublicationNo. WO 2007/061742); Lip2: Lip2 terminator sequence from Yarrowia Lip2gene (GenBank Accession No. AJ012632) ClaI/EcoRI LoxP::Ura3::LoxP,comprising: (11385-9648) LoxP sequence (SEQ ID NO: 320); Yarrowia Ura3gene (GenBank Accession No. AJ306421); LoxP sequence (SEQ ID NO: 320)EcoRI/PacI EXP1::FmD12S::ACO, comprising: (9648-6951) EXP1: Yarrowialipolytica export protein (EXP1) promoter (labeled as “Exp” in Figure;U.S. patent application No. 11/265761); FmD12S: codon-optimized delta-12elongase (SEQ ID NO: 346), derived from Fusarium moniliforme (labeled as“FD12S” in Figure; PCT Publication No. WO 2005/047485); Aco: Acoterminator sequence from Yarrowia Aco gene (GenBank Accession No.AJ001300)

The pZKL1-2SP98C plasmid was digested with AscI/SphI and then used fortransformation of strain Y4217U2 according to the General Methods. Thetransformed cells were plated onto MM plates, and plates were maintainedat 30° C. for 3 to 4 days. Single colonies were re-streaked onto MMplates, and the resulting colonies were then used to inoculate liquidMM. The liquid cultures were then shaken at 250 rpm/min for 2 days at30° C. The cells were collected by centrifugation, resuspended in HGM,and then shaken at 250 rpm/min for 5 days. The cells were collected bycentrifugation, and lipids were extracted. Fatty acid methyl esters wereprepared by trans-esterification and subsequently analyzed with aHewlett-Packard 6890 GC.

GC analyses showed that most of the selected 72 strains produced 40-44%EPA of total lipids. Six strains, designated as Y4259, Y4260, Y4261,Y4262, Y4263, and Y4264, produced about 46.5%, 44.5%, 44.5%, 44.8%,44.5%, and 44.3% EPA of total lipids, respectively.

The final genotype of strain Y4259 with respect to wild type Yarrowialipolytica ATCC #20362 was: YALI0C18711g-, Pex10-, YALI0F24167g-,unknown 1-, unknown 3-, unknown 8-, GPD::FmD12::Pex20, YAT1::FmD12::OCT,GPM/FBAIN::FmD12S::OCT, EXP1::FmD12S::Aco, YAT1::ME3S::Pex16,EXP1::ME3S::Pex20 (2 copies), GPAT::EgD9e::Lip2, EXP1::EgD9eS::Lip1,FBAINm::EgD9eS::Lip2, FBA::EgD9eS::Pex20, GPD::EgD9eS::Lip2,YAT1::EgD9eS::Lip2, FBAINm::EgD8M::Pex20, FBAIN::EgD8M::Lip1 (2 copies),EXP1::EgD8M::Pex16, GPDIN::EgD8M::Lip1, YAT1::EgD8M::Aco,FBAIN::EgD5::Aco, EXP1::EgD5S::Pex20, YAT1::EgD5S::Aco, YAT1::RD5S::OCT,YAT1::PaD17S::Lip1, EXP1::PaD17::Pex16, FBAINm::PaD17::Aco,YAT1::YICPT1::ACO, GPD::YICPT1::ACO.

Generation of Strain Y4259U2 (Ura3-):

In order to disrupt the Ura3 gene in Y4259 strain, construct pZKUM (FIG.50A; SEQ ID NO:353) was used to integrate a Ura3 mutant gene into theUra3 gene of strain Y4259. The plasmid pZKUM contained the followingcomponents:

TABLE 18 Description of Plasmid pZKUM (SEQ ID NO: 353) RE Sites AndNucleotides Within SEQ Description Of Fragment And Chimeric Gene ID NO:353 Components SalI/PacI Synthetic mutant Ura3 gene (SEQ ID NO: 354,wherein (32845-1) the 1459 bp DNA fragment contains a 33 bp deletionfrom +21 to +53, a 1 bp deletion at +376 and a 3 bp deletion from +400to +403 of the Yarrowia Ura3 coding region (GenBank Accession No.AJ306421)) 1112-232 ColE1 plasmid origin of replication 2042-1182Ampicillin-resistance gene (Amp^(R)) for selection in E. coli

A total of 3 transformants grown on MM+5-FOA plates were picked andre-streaked onto MM plates and MM+5-FOA plates. All 3 strains had aUra−phenotype (i.e., cells could grow on MM+5-FOA plates, but not on MMplates). The cells were scraped from the MM+5-FOA plates, and lipidswere extracted. Fatty acid methyl esters were prepared bytrans-esterification and subsequently analyzed with a Hewlett-Packard6890 GC.

GC analyses showed the presence of 31.4%, 31% and 31.3% EPA in the #1,#2 and #3 transformants with pZKUM grown on MM+5-FOA plates. These threestrains were designated as strains Y4259U1, Y4259U2 and Y4259U3,respectively (collectively, Y4259U).

Generation of Y4305 Strain to Produce about 53% EPA of Total Lipids:

Construct pZKD2-5U89A2 (FIG. 50B; SEQ ID NO:355) was generated tointegrate one delta-9 elongase gene, one delta-5 desaturase gene, onedelta-8 desaturase gene, and one delta-12 desaturase gene into thediacylglycerol acyltransferase (DGAT2) loci of strain Y4259U2, tothereby enable higher level production of EPA. The pZKD2-5U89A2 plasmidcontained the following components:

TABLE 19 Description of Plasmid pZKD2-5U89A2 (SEQ ID NO: 355) RE SitesAnd Nucleotides Within SEQ Description Of Fragment And Chimeric Gene IDNO: 355 Components AscI/BsiWI 728 bp 5′ portion of Yarrowia DGAT2 gene(SEQ ID (1-736) NO: 356) (labeled as “YLDGAT5′” in Figure; U.S. Pat. No.7,267,976) PacI/SphI 714 bp 3′ portion of Yarrowia DGAT2 gene (SEQ ID(4164-3444) NO: 356) (labeled as “YLDGAT3′” in Figure; U.S. Pat. No.7,267,976) SwaI/BsiWI YAT1::FmD12S::Lip2, comprising: (13377-1) YAT1:Yarrowia lipolytica YAT1 promoter (labeled as “YAT” in Figure; PatentPublication US 2006/0094102-A1); FmD12S: codon-optimized delta-12elongase (SEQ ID NO: 346), derived from Fusarium moniliforme (labeled as“F.D12S” in Figure; PCT Publication No. WO 2005/047485); Lip2: Lip2terminator sequence from Yarrowia Lip2 gene (GenBank Accession No.AJ012632) PmeI/SwaI FBAIN::EgD8M::Lip1 comprising: (10740-13377) FBAIN:Yarrowia lipolytica FBAIN promoter (U.S. Pat. No. 7,202,356); EgD8M:Synthetic mutant delta-8 desaturase (SEQ ID NO: 327; U.S. patentapplication No. 11/635258), derived from Euglena gracilis (“EgD8S”; U.S.Pat. No. 7,256,033); Lip1: Lip1 terminator sequence from Yarrowia Lip1gene (GenBank Accession No. Z50020) ClaI/PmeI YAT1::E389D9eS::OCT,comprising: (8846-10740) YAT1: Yarrowia lipolytica YAT1 promoter(labeled as “YAT” in Figure; Patent Publication US 2006/0094102-A1);E389D9eS: codon-optimized delta-9 elongase (SEQ ID NO: 358), derivedfrom Eutreptiella sp. CCMP389 (labeled as “D9ES-389” in Figure; PCTPublication No. WO 2007/061742); OCT: OCT terminator sequence fromYarrowia OCT gene (GenBank Accession No. X69988) ClaI/EcoRI YarrowiaUra3 gene (GenBank Accession No. (8846-6777) AJ306421) EcoRI/PacIEXP1::EgD5S::ACO, comprising: (6777-4164) EXP1: Yarrowia lipolyticaexport protein (EXP1) promoter (labeled as “Exp” in Figure; U.S. patentapplication No. 11/265761); EgD5S: codon-optimized delta-5 desaturase(SEQ ID NO: 332), derived from Euglena gracilis (Patent Publication US2007-0292924-A1); Aco: Aco terminator sequence from Yarrowia Aco gene(GenBank Accession No. AJ001300)

The pZKD2-5U89A2 plasmid was digested with AscI/SphI and then used fortransformation of strain Y4259U2 according to the General Methods. Thetransformed cells were plated onto MM plates, and plates were maintainedat 30° C. for 3 to 4 days. Single colonies were re-streaked onto MMplates, and the resulting colonies were used to inoculate liquid MM.Liquid cultures were shaken at 250 rpm/min for 2 days at 30° C. Thecells were collected by centrifugation, resuspended in HGM, and thenshaken at 250 rpm/min for 5 days. The cells were collected bycentrifugation, and lipids were extracted. Fatty acid methyl esters wereprepared by trans-esterification and subsequently analyzed with aHewlett-Packard 6890 GC.

GC analyses showed that most of the selected 96 strains produced 40-46%EPA of total lipids. Four strains, designated as Y4305, Y4306, Y4307 andY4308, produced about 53.2%, 46.4%, 46.8%, and 47.8% EPA of totallipids, respectively. The complete lipid profile of Y4305 is as follows:16:0 (2.8%), 16:1 (0.7%), 18:0 (1.3%), 18:1 (4.9%), 18:2 (17.6%), ALA(2.3%), EDA (3.4%), DGLA (2.0%), ARA (0.6%), ETA (1.7%), and EPA(53.2%). The total lipid % dry cell weight (dcw) was 27.5.

The final genotype of strain Y4305 with respect to wild type Yarrowialipolytica ATCC #20362 was SCP2- (YALI0E01298g), YALI0C18711g-, Pex10-,YALI0F24167g-, unknown 1-, unknown 3-, unknown 8-, GPD::FmD12::Pex20,YAT1::FmD12::OCT, GPM/FBAIN::FmD12S::OCT, EXP1::FmD12S::Aco,YAT1::FmD12S::Lip2, YAT1::ME3S::Pex16, EXP1::ME3S::Pex20 (3 copies),GPAT::EgD9e::Lip2, EXP1::EgD9eS::Lip1, FBAINm::EgD9eS::Lip2,FBA::EgD9eS::Pex20, GPD::EgD9eS::Lip2, YAT1::EgD9eS::Lip2,YAT1::E389D9eS::OCT, FBAINm::EgD8M::Pex20, FBAIN::EgD8M::Lip1 (2copies), EXP1::EgD8M::Pex16, GPDIN::EgD8M::Lip1, YAT1::EgD8M::Aco,FBAIN::EgD5::Aco, EXP1::EgD5S::Pex20, YAT1::EgD5S::Aco,EXP1::EgD5S::ACO, YAT1::RD5S::OCT, YAT1::PaD17S::Lip1,EXP1::PaD17::Pex16, FBAINm::PaD17::Aco, YAT1::YICPT1::ACO,GPD::YICPT1::ACO.

Generation of Strain Y4305U3 (Ura3-):

In order to disrupt the Ura3 gene in strain Y4305, construct pZKUM (FIG.50A; SEQ ID NO:353) was used to integrate a Ura3 mutant gene into theUra3 gene of strain Y4305. A total of 8 transformants grown on MM+5-FOAplates were picked and re-streaked onto MM plates and MM+5-FOA plates,separately. All 8 strains had a Ura− phenotype (i.e., cells could growon MM+5-FOA plates, but not on MM plates). The cells were scraped fromthe MM+5-FOA plates, and lipids were extracted. Fatty acid methyl esterswere prepared by trans-esterification and subsequently analyzed with aHewlett-Packard 6890 GC.

GC analyses showed the presence of 37.6%, 37.3% and 36.5% EPA in pZKUMtransformants #1, #6 and #7 grown on MM+5-FOA plates. These threestrains were designated as strains Y4305U1, Y4305U2 and Y4305U3,respectively (collectively, Y4305U).

Construction of Yarrowia lipolytica Strain Y4184U

Y. lipolytica strain Y4184U was used as the host in Examples 32, 33, 34and 51, infra. Strain Y4184U was derived from Y. lipolytica ATCC #20362,and is capable of producing about 31% EPA relative to the total lipidsvia expression of a delta-9 elongase/delta-8 desaturase pathway.

The development of strain Y4184U required the construction of strainY2224, strain Y4001, strain Y4001U, strain Y4036, strain Y4036U andstrain Y4069 (supra). Further development of strain Y4184U (diagrammedin FIG. 51A) required construction of strain Y4084 (producing 14% EPA),strain Y4084U1 (Ura−), strain Y4127 (producing 18% EPA), strain Y4127U2(Ura−), strain Y4158 (producing 25% EPA), strain Y4158U1 (Ura−), andstrain 4184 (producing 30.7% EPA). Although the details concerningtransformation and selection of the EPA-producing strains developedafter strain Y4069 will not be elaborated herein, the methodology usedfor isolation of strain Y4084, strain Y4084U1, strain Y4127, strainY4127U2, strain Y4158, strain Y4158U1, strain Y4184, and strain Y4184Uwas as described during construction of strain Y4305, supra.

Briefly, construct pZP3-Pa777U (FIG. 47A; SEQ ID NO:338) was utilized tointegrate three delta-17 desaturase genes into the Pox3 loci (GenBankAccession No. AJ001301) of strain Y4069, thereby resulting in isolationof strain Y4084 (producing 14% EPA). Strain Y4084U1 was created viatemporary expression of the Cre recombinase enzyme in construct pY117(FIG. 47B; SEQ ID NO:343) within strain Y4084 to produce a Ura−phenotype. Construct pZP2-2988 (FIG. 48A; SEQ ID NO:345) was thenutilized to integrate one delta-12 desaturase gene, two delta-8desaturase genes, and one delta-9 elongase gene into the Pox2 loci(GenBank Accession No. AJ001300) of strain Y4084U1, thereby resulting inisolation of strain Y4127 (producing 18% EPA). Yarrowia lipolyticastrain Y4127 was deposited with the American Type Culture Collection onNov. 29, 2007 and bears the designation ATCC PTA-8802.

Strain Y4127U2 was created by disrupting the Ura3 gene in strain Y4127via construct pZKUE3S (FIG. 48B; SEQ ID NO:351), comprising a chimericEXP1::ME3S::Pex20 gene targeted for the Ura3 gene. ConstructpZKL1-2SP98C (FIG. 49B; SEQ ID NO:352) was utilized to integrate onedelta-9 elongase gene, one delta-8 desaturase gene, one delta-12desaturase gene, and one Yarrowia lipolytica diacylglycerolcholinephosphotransferase gene (CPT1) into the Lip1 loci (GenBankAccession No. Z50020) of strain Y4127U2, thereby resulting in isolationof strain Y4158 (producing 25% EPA). A Ura− derivative (i.e., strainY4158U1) was then created, via transformation with construct pZKUE3S(FIG. 48B; SEQ ID NO:351), comprising a chimeric EXP1::ME3S::Pex20 genetargeted for the Ura3 gene. Finally, construct pZKL2-5U89GC (FIG. 49A;SEQ ID NO:348) was utilized to integrate one delta-9 elongase gene, onedelta-8 desaturase gene, one delta-5 desaturase gene, and one Yarrowialipolytica CPT1 into the Lip2 loci (GenBank Accession No. AJ012632) ofY4158U1, thereby resulting in isolation of strain Y4184.

The complete lipid profile of strain Y4184 is as follows: 16:0 (3.1%),16:1 (1.5%), 18:0 (1.8%), 18:1 (8.7%), 18:2 (31.5%), ALA (4.9%), EDA(5.6%), DGLA (2.9%), ARA (0.6%), ETA (2.4%), and EPA (28.9%). The totallipid % dry cell weight (dcw) was 23.9.

The final genotype of strain Y4184 with respect to wildtype Yarrowialipolytica ATCC #20362 was unknown 1-, unknown 2-, unknown 4-, unknown5-, unknown 6-, unknown 7-, YAT1::ME3S::Pex16, EXP1::ME3S::Pex20 (2copies), GPAT::EgD9e::Lip2, FBAINm::EgD9eS::Lip2, EXP1::EgD9eS::Lip1,FBA::EgD9eS::Pex20, YAT1::EgD9eS::Lip2, GPD::EgD9eS::Lip2,GPDIN::EgD8M::Lip1, YAT1::EgD8M::Aco, EXP1::EgD8M::Pex16,FBAINm::EgD8M::Pex20, FBAIN::EgD8M::Lip1 (2 copies),GPM/FBAIN::FmD12S::Oct, EXP1::FmD12S::Aco, YAT1::FmD12::Oct,GPD::FmD12::Pex20, EXP1::EgD5S::Pex20, YAT1::EgD5S::Aco,YAT1::Rd5S::Oct, FBAIN::EgD5::Aco, FBAINm::PaD17::Aco,EXP1::PaD17::Pex16, YAT1::PaD17S::Lip1, YAT1::YICPT1::Aco,GPD::YICPT1::Aco.

In order to disrupt the Ura3 gene in strain Y4184, construct pZKUM (FIG.50A; SEQ ID NO:353) was used to integrate a Ura3 mutant gene into theUra3 gene of strain Y4184.

A total of 11 transformants grown on MM+5-FOA plates were picked andre-streaked onto MM plates and MM+5-FOA plates, separately. All 11strains had a Ura− phenotype (i.e., cells could grow on MM+5-FOA plates,but not on MM plates). The cells were scraped from the MM+5-FOA plates;lipids were extracted; and fatty acid methyl esters were prepared bytrans-esterification and subsequently analyzed with a Hewlett-Packard6890 GC.

GC analyses showed the presence of 11.2%, 10.6%, and 15.5% EPA in the#7, #8 and #10 transformants with pZKUM grown on MM+5-FOA plates. Thesethree strains were designated as strains Y4184U1, Y4184U2 and Y4184U4,respectively (collectively, Y4184U).

Example 1 Euglena gracilis Growth Conditions, Lipid Profile and mRNAIsolation

Euglena gracilis was obtained from Dr. Richard Triemer's lab at MichiganState University (East Lansing, Mich.). From 10 mL of actively growingculture, a 1 mL aliquot was transferred into 250 mL of Euglena gracilis(Eg) Medium in a 500 mL glass bottle. Eg medium was made by combining 1gof sodium acetate, 1g of beef extract (Cat. No. U126-01, DifcoLaboratories, Detroit, Mich.), 2g of Bacto® tryptone (0123-17-3, DifcoLaboratories), and 2g of Bacto® yeast extract (Cat. No. 0127-17-9, DifcoLaboratories) in 970 mL of water. After filter sterilizing, 30 mL ofsoil-water supernatant (Cat. No. 15-3790, Carolina Biological SupplyCompany, Burlington, N.C.) were aseptically added to produce the finalEg medium. Euglena gracilis cultures were grown at 23° C. with a 16 hlight, 8 h dark cycle for 2 weeks with no agitation.

After 2 weeks, 10 mL of culture were removed for lipid analysis andcentrifuged at 1,800×g for 5 min. The pellet was washed once with waterand re-centrifuged. The resulting pellet was dried for 5 min undervacuum, resuspended in 100 μL of trimethylsulfonium hydroxide (TMSH),and incubated at room temperature for 15 min with shaking. After this,0.5 mL of hexane were added, and the vials were incubated for 15 min atroom temperature with shaking. Fatty acid methyl esters (5 μL injectedfrom hexane layer) were separated and quantified using a Hewlett-Packard6890 Gas Chromatograph fitted with an Omegawax 320 fused silicacapillary column (Supelco Inc., Cat. No. 24152). The oven temperaturewas programmed to hold at 220° C. for 2.7 min, increase to 240° C. at20° C./min, and then hold for an additional 2.3 min. Carrier gas wassupplied by a Whatman hydrogen generator. Retention times were comparedto those for methyl esters of standards commercially available (Nu-ChekPrep, Inc. Cat. No. U-99-A), and the resulting chromatogram is shown inFIG. 27.

The remaining 2 week culture (240 mL) was pelleted by centrifugation at1,800×g for 10 min, washed once with water, and re-centrifuged. TotalRNA was extracted from the resulting pellet using the RNA STAT-60™reagent (TEL-TEST, Inc., Friendswood, Tex.) and following themanufacturer's protocol provided (use 5 mL of reagent, dissolved RNA in0.5 mL of water). In this way, 1 mg of total RNA (2 mg/mL) was obtainedfrom the pellet. The mRNA was isolated from 1 mg of total RNA using themRNA Purification Kit (Amersham Biosciences, Piscataway, N.J.) followingthe manufacturer's protocol provided. In this way, 85 μg of mRNA wereobtained.

Example 2 Euglena gracilis cDNA Synthesis, Library Construction andSequencing

A cDNA library was generated using the Cloneminer™ cDNA LibraryConstruction Kit (Cat. No. 18249-029, Invitrogen Corporation, Carlsbad,Calif.) and following the manufacturer's protocol provided (Version B,25-0608). Using the non-radiolabeling method, cDNA was synthesized from3.2 μg of mRNA (described above) using the Biotin-attB2-Oligo(dT)primer. After synthesis of the first and second strand, the attB1adapter was added; ligation was performed; and the cDNA was sizefractionated using column chromatography. DNA from fractions 7 and 8(size ranging from ˜800-1500 bp) were concentrated, recombined intopDONR™ 222, and transformed into E. coli ElectroMAX™ DH10B™ T1Phage-Resistant cells (Invitrogen Corporation). The Euglena gracilislibrary was named eeg1c.

For sequencing, clones first were recovered from archived glycerolcultures grown/frozen in 384-well freezing media plates. Using anautomatic QPix colony picker (Genetix), cells were picked and then usedto inoculate 96-well deep-well plates containing LB+50 μg/mL kanamycin.After growing 20 h at 37° C., cells were pelleted by centrifugation andstored at −20° C. Plasmids then were isolated on an Eppendorf 5Primerobot, using a modified 96-well format alkaline lysis miniprep method(Eppendorf PerfectPrep). Briefly, a filter and vacuum manifold were usedto facilitate removal of cellular debris after acetate precipitation.Plasmid DNA was then bound on a second filter plate directly from thefiltrate, washed, dried, and eluted.

Plasmids were end-sequenced in 384-well plates, using vector-primed M13FUniversal primer (SEQ ID NO:1) and the ABI BigDye version 3 Prismsequencing kit. For the sequencing reaction, 100-200 ng of template and6.4 pmol of primer were used, and the following reaction conditions wererepeated 25 times: 96° C. for 10 sec, 50° C. for 5 sec and 60° C. for 4min. After ethanol-based cleanup, cycle sequencing reaction productswere resolved and detected on Perkin-Elmer ABI 3700 automatedsequencers.

Example 3 Identification of C20-PUFA Elongating Enzyme Homologs fromEuglena gracilis cDNA Library eeg1c

cDNA clones encoding C20-PUFA elongating enzyme homologs (i.e.,“C20-PUFA Elo”) were identified by conducting BLAST (Basic LocalAlignment Search Tool; Altschul et al., J. Mol. Biol. 215:403-410(1993)) searches for similarity to sequences contained in the BLAST “nr”database (comprising all non-redundant GenBank CDS translations,sequences derived from the 3-dimensional structure Brookhaven ProteinData Bank, the last major release of the SWISS-PROT protein sequencedatabase, EMBL and DDBJ databases). The cDNA sequences obtained inExample 2 were analyzed for similarity to all publicly available DNAsequences contained in the “nr” database using the BLASTN algorithmprovided by the National Center for Biotechnology Information (NCBI).The DNA sequences were translated in all reading frames and compared forsimilarity to all publicly available protein sequences contained in the“nr” database using the BLASTX algorithm (Gish and States, Nat. Genet.3:266-272 (1993)) provided by the NCBI. For convenience, the P-value(probability) of observing a match of a cDNA sequence to a sequencecontained in the searched databases merely by chance as calculated byBLAST are reported herein as “pLog” values, which represent the negativeof the logarithm of the reported P-value. Accordingly, the greater thepLog value, the greater the likelihood that the cDNA sequence and theBLAST “hit” represent homologous proteins.

The BLASTX search using the nucleotide sequences from cloneeeg1c.pk005.p14.f revealed similarity of the protein encoded by the cDNAto the C20-PUFA Elo from Pavlova sp. CCMP459 (SEQ ID NO:2) (NCBIAccession No. AAV33630 (GI 54307108), locus AAV33630, CDS AY630573;Pereira et al., Biochem. J. 384:357-366 (2004)). The sequence of aportion of the cDNA insert from clone eeg1c.pk005.p14.f is shown in SEQID NO:3 (5′ end of cDNA insert). Subsequently, the full insert sequence(i.e., eeg1c.pk005.p14.f:fis) was obtained and is shown in SEQ ID NO:4.Sequence for the coding sequence (CDS) is shown in SEQ ID NO:5. Sequencefor the corresponding deduced amino acid sequence is shown in SEQ IDNO:6.

Full insert sequencing (FIS) was carried out using a modifiedtransposition protocol. Clones identified for FIS were recovered fromarchived glycerol stocks as single colonies, and plasmid DNA wasisolated via alkaline lysis. Plasmid templates were transposed via theTemplate Generation System (TGS II) transposition kit (Finnzymes Oy,Espoo, Finland), following the manufacturer's protocol. The transposedDNA was transformed into EH10B electro-competent cells (Edge BioSystems,Gaithersburg, Md.) via electroporation. Multiple transformants wererandomly selected from each transposition reaction, plasmid DNA wasprepared, and templates were sequenced as above (ABI BigDye v3.1)outward from the transposition event site, utilizing unique primers SeqE(SEQ ID NO:7) and SeqW (SEQ ID NO:8).

Sequence data was collected (ABI Prism Collections software) andassembled using the Phrap sequence assembly program (P. Green,University of Washington, Seattle). Assemblies were viewed by the Consedsequence editor (D. Gordon, University of Washington, Seattle) for finalediting.

The amino acid sequence set forth in SEQ ID NO:6 was evaluated byBLASTP, yielding a pLog value of 61.22 (E value of 6e-62) versus thePavlova sp. CCMP459 C20-PUFA Elo (SEQ ID NO:2). The amino acid sequenceset forth in SEQ ID NO:6 is 45.1% identical to the Pavlova sp. CCMP459C20-PUFA Elo sequence (SEQ ID NO:2) using the Jotun Hein method.Sequence percent identity calculations performed by the Jotun Heinmethod (Hein, J. J., Meth. Enz. 183:626-645 (1990)) were done using theMegAlign™ v6.1 program of the LASERGENE bioinformatics computing suite(DNASTAR Inc., Madison, Wis.) with the default parameters for pairwisealignment (KTUPLE=2). The amino acid sequence set forth in SEQ ID NO:6is 40.4% identical to the Pavlova sp. CCMP459 C20-PUFA Elo sequence (SEQID NO:2) using the Clustal V method. Sequence percent identitycalculations performed by the Clustal V method (Higgins, D. G. andSharp, P. M., Comput. Appl. Biosci. 5:151-153 (1989); Higgins et al.,Comput. Appl. Biosci. 8:189-191 (1992)) were done using the MegAlign™v6.1 program of the LASERGENE bioinformatics computing suite (supra)with the default parameters for pairwise alignment (KTUPLE=1, GAPPENALTY=3, WINDOW=5 and DIAGONALS SAVED=5 and GAP LENGTH PENALTY=10).BLAST scores and probabilities indicate that the instant nucleic acidfragment (SEQ ID NO:5) encodes an entire Euglena gracilis C20-PUFA Elogene, hereby named EgC20elo1.

FIG. 25 summarizes BLASTP and percent identity values for EgC20elo1(Example 3), EgDHAsyn1 (Example 4, infra), and EgDHAsyn2 (Example 5,infra).

Example 4 Identification of DHA Synthase 1 (EgDHAsyn1) from Euglenagracilis cDNA Library eeg1c

cDNA clones encoding additional C20-PUFA Elo homologs were identified byconducting BLAST searches for similarity to sequences contained in theBLAST “nr” database as described in Example 3.

The BLASTX search using the nucleotide sequences from cloneeeg1c.pk016.e6.f (also called pKR1049) revealed similarity of theprotein encoded by the cDNA to the C20-PUFA Elo from Pavlova sp. CCMP459(SEQ ID NO:2) (NCBI Accession No. AAV33630 (GI 54307108), locusAAV33630, CDS AY630573; Pereira et al., Biochem. J. 384:357-366 (2004)).The sequence of a portion of the cDNA insert from clone eeg1c.pk016.e6.fis shown in SEQ ID NO:9 (5′ end of cDNA insert). Subsequently, the fullinsert sequence (eeg1c.pk016.e6.f:fis) was obtained as described inExample 3 and is shown in SEQ ID NO:10. The coding sequence is shown inSEQ ID NO:11; the corresponding deduced amino acid sequence is shown inSEQ ID NO:12.

The amino acid sequence set forth in SEQ ID NO:12 was evaluated byBLASTP as described in Example 3. Interestingly, SEQ ID NO:12 was foundto be similar to both C20-PUFA Elo and delta-4 fatty acid desaturase.The N-terminus of SEQ ID NO:12 (from approximately amino acids 16-268)yields a pLog value of 60.30 (E value of 5e-61; 124/258 identical aminoacids; 48% identity) versus the Pavlova sp. CCMP459 C20-PUFA Elo (SEQ IDNO:2). The C-terminus of SEQ ID NO:12 (from approximately amino acids253-793) yields an E value of 0.0 (535/541 identical amino acids; 98%identity), versus the delta-4 fatty acid desaturase from Euglenagracilis (SEQ ID NO:13) (NCBI Accession No. AAQ19605 (GI 33466346),locus AAQ19605, CDS AY278558; Meyer et al., Biochemistry42(32):9779-9788 (2003)). BLAST scores and probabilities indicate thatthe instant nucleic acid fragment (SEQ ID NO:11) encodes an entireEuglena gracilis C20-PUFA Elo/delta-4 fatty acid desaturase fusion gene,hereby named Euglena gracilis DHA synthase 1 (EgDHAsyn1).

The amino acid sequence of EgDHAsyn1 (SEQ ID NO:12) is 47.8% identicalto the C20-PUFA Elo from Pavlova sp. CCMP459 (SEQ ID NO:2) and 98.9%identical to the delta-4 fatty acid desaturase from Euglena gracilis(SEQ ID NO:13), using the Jotun Hein method as described in Example 3.The amino acid sequence of EgDHAsyn1 (SEQ ID NO:12) is 41.2% identicalto the C20-PUFA Elo from Pavlova sp. CCMP459 (SEQ ID NO:2) and 98.9%identical to the delta-4 fatty acid desaturase from Euglena gracilis(SEQ ID NO:13), using the Clustal V method as described in Example 3.

FIG. 27 summarizes BLASTP and percent identity values for EgDHAsyn1(Example 4), EgC20elo1 (Example 3, supra) and EgDHAsyn2 (Example 5,infra).

Example 5 Identification of DHA synthase 2 (EgDHAsyn2) from Euglenagracilis cDNA Library eeg1c

Approximately 17,000 clones of the Euglena gracilis cDNA library eeg1cwere plated onto three large square (24 cm×24 cm) petri plates (Corning,Corning, N.Y.) each containing LB+50 μg/mL kanamycin agar media. Cellswere grown overnight at 37° C., and plates were then cooled to roomtemperature.

Colony Lifts:

Biodyne B 0.45 μm membrane (Cat. No. 60207, Pall Corporation, Pensacola,Fla.) was trimmed to approximately 22 cm×22 cm, and the membrane wascarefully laid on top of the agar to avoid air bubbles. After incubationfor 2 min at room temperature, the membrane was marked for orientation,lifted off with tweezers, and placed colony-side up on filter papersoaked with 0.5 M sodium hydroxide and 1.5 M sodium chloride. Afterdenaturation for 4 min, the sodium hydroxide was neutralized by placingthe membrane on filter paper soaked with 0.5 M Tris-HCL (pH 7.5) and 1.5M sodium chloride for 4 min. This step was repeated, and the membranewas rinsed briefly in 2×SSC buffer (20×SSC is 3M sodium chloride, 0.3 Msodium citrate; pH 7.0) and air dried on filter paper.

Hybridization:

Membranes were pre-hybridized at 65° C. in 200 mL hybridization solutionfor 2 hr. Hybridization solution contained 6×SSPE (20×SSPE is 3 M sodiumchloride, 0.2 M sodium phosphate, 20 mM EDTA; pH 7.4), 5×Denhardt'sreagent (100×Denhardt's reagent is 2% (w/v) Ficoll, 2% (w/v)polyvinylpyrrolidone, 2% (w/v) acetylated bovine serum albumin), 0.5%sodium dodecyl sulfate (SDS), 100 μg/mL sheared salmon sperm DNA, and 5%dextran sulfate.

A DNA probe was made using an agarose gel purified NcoI/NotI DNAfragment, containing EgDHAsyn1*, from pY141 (described in Example 10herein) labeled with P³² dCTP using the RadPrime DNA Labeling System(Cat. No. 18428-011, Invitrogen, Carlsbad, Calif.), following themanufacturer's instructions. Unincorporated P³² dCTP was separated usinga NICK column (Cat. No. 17-0855-02, Amersham Biosciences, Piscataway,N.J.), following the manufacturer's instructions. The probe wasdenatured for 5 min at 100° C. and placed on ice for 3 min; then, halfwas added to the hybridization solution.

The membrane was hybridized with the probe overnight at 65° C. withgentle shaking and then washed the following day twice with 2×SSCcontaining 0.5% SDS (5 min each) and twice with 0.2×SSC containing 0.1%SDS (15 min each). After washing, hyperfilm (Cat. No. RPN30K, AmershamBiosciences) was exposed to the membrane overnight at −80° C.

Based on alignment of plates with the exposed hyperfilm, positivecolonies were picked using the blunt end of a Pasteur pipette into 1 mLof water and then vortexed. Several dilutions were made and plated ontosmall round Petri dishes (82 mm) containing LB media plus 50 μg/mLkanamycin to obtain around 100 well isolated colonies on a single plate.Lifts were done as described above except NytranN membrane circles (Cat,No. 10416116, Schleicher & Schuell, Keene, N.H.) were used, andhybridization was carried out in 100 mL using the remaining radiolabeledprobe. In this way, one positive clone was identified (designatedeeg1c-1). The plasmid from eeg1c-1 may also be referred to as pLF116.

The individual positive clone was grown at 37° C. in LB+50 μg/mLkanamycin liquid media, and plasmid was purified using the QIAprep® SpinMiniprep Kit (Qiagen Inc., Valencia, Calif.) following themanufacturer's protocol. The plasmid insert was sequenced as describedin Example 2, with the ABI Big Dye version 3 Prism sequencing kit usingvector-primed M13F Universal primer (SEQ ID NO:1), vector-primed M13revprimer (SEQ ID NO:14), and the poly(A) tail-primed WobbleToligonucleotides. Briefly, the WobbleT primer is an equimolar mix of 21mer poly(T)A, poly(T)C, and poly(T)G, used to sequence the 3′ end ofcDNA clones. Based on initial sequence data, additional internalfragment sequence was obtained in a similar way using oligonucleotidesoEUGel4-1 (SEQ ID NO:15), EgEloD4Mut-5 (SEQ ID NO:16), oEUGel4-2 (SEQ IDNO:17), EgDHAsyn5′ (SEQ ID NO:18), and EgDHAsyn3′ (SEQ ID NO:19). Inthis way, the full insert sequence of eeg1c-1 was obtained and is shownin SEQ ID NO:20. The coding sequence is shown as SEQ ID NO:21, while thecorresponding deduced amino acid sequence is shown as SEQ ID NO:22.

The amino acid sequence set forth in SEQ ID NO:22 was evaluated byBLASTP as described in Example 3. As was the case for EgDHAsyn1, SEQ IDNO:22 was also found to be similar to both C20-PUFA Elo and delta-4fatty acid desaturase. The N-terminus of SEQ ID NO:22 (fromapproximately amino acids 41-268) yields a pLog value of 61.0 (E valueof 1 e-61; 118/231 identical amino acids; 51% identity) versus thePavlova sp. CCMP459 C20-PUFA Elo (SEQ ID NO:2). The C-terminus of SEQ IDNO:22 (from approximately amino acids 253-793) yields an E value of 0.0(541/541 identical amino acids; 100% identity), versus the amino acidsequence of delta-4 fatty acid desaturase from Euglena gracilis (SEQ IDNO:13). BLAST scores and probabilities indicate that the instant nucleicacid fragment (SEQ ID NO:21) encodes an entire Euglena gracilis C20-PUFAElo/delta-4 fatty acid desaturase fusion gene, hereby named Euglenagracilis DHA synthase 2 (EgDHAsyn2).

The amino acid sequence of EgDHAsyn2 (SEQ ID NO:22) is 48.2% identicalto the C20-PUFA Elo from Pavlova sp. CCMP459 (SEQ ID NO:2) and 100%identical to the delta-4 fatty acid desaturase from Euglena gracilis(SEQ ID NO:13), using the Jotun Hein method as described in Example 3.The amino acid sequence of EgDHAsyn2 (SEQ ID NO:22) is 41.2% identicalto the C20-PUFA Elo from Pavlova sp. CCMP459 (SEQ ID NO:2) and 100%identical to the delta-4 fatty acid desaturase from Euglena gracilis(SEQ ID NO:13), using the Clustal V method as described in Example 3.

FIG. 25 summarizes BLASTP and percent identity values for EgDHAsyn2(Example 5), EgC20elo1 (Example 3, supra) and EgDHAsyn1 (Example 4,supra).

Example 6 Primary Structure Analysis of EqC20elo1, EgDHAsyn1 andEgDHAsyn2

Given the 100% amino acid identity between the C-terminus of EgDHAsyn2(SEQ ID NO:22) and the Euglena gracilis delta-4 desaturase (SEQ IDNO:13), a nucleotide sequence alignment was carried out between thecoding sequence of EgDHAsyn2 (SEQ ID NO:21), the cDNA sequence of theEuglena gracilis delta-4 desaturase (SEQ ID NO:23) (NCBI Accession No.AY278558 (GI 33466345), locus AY278558, Meyer et al., Biochemistry42(32):9779-9788 (2003)), and the coding sequence of the Euglenagracilis delta-4 desaturase (SEQ ID NO:24) (Meyer et al., supra).Sequence alignment was performed by the Clustal W method (using theMegAlign™ v6.1 program of the LASERGENE bioinformatics computing suite(DNASTAR Inc.) with the default parameters for multiple alignment (GAPPENALTY=10, GAP LENGTH PENALTY=0.2, Delay Divergen Seqs(%)=30, DNATransition Weight=0.5, Protein Weight Matrix=Gonnet Series, DNA WeightMatrix=IUB). The alignment is shown in FIG. 2. The Euglena gracilisdelta-4 desaturase coding sequence is named EgD4_CDS (SEQ ID NO:24); theEuglena gracilis delta-4 desaturase cDNA sequence is named EgD4_cDNA(SEQ ID NO:23); and the Euglena gracilis DHA synthase 2 coding sequenceis named EgDHAsyn2_CDS (SEQ ID NO:21).

The 5′ end (where the sequences are divergent) and the 3′ end (where thesequences are identical) of the alignment are truncated in order to fitthe alignment on one page. FIG. 2 illustrates that the sequences arehighly divergent from the start of the Euglena gracilis delta-4desaturase cDNA to 83 by upstream of the coding sequence (CDS) startsite. It is clear from the alignment that the nucleotide sequences forEgD4_cDNA and EgDHAsyn2_CDS are identical from 83 by upstream of the CDSstart site of the Euglena gracilis delta-4 desaturase cDNA sequence (SEQID NO:23), which is equivalent to nucleotide 674 of the EgDHAsyn2_CDS(SEQ ID NO:21), through to the end of the sequences. At the exact pointof divergence, a NotI site can be found in the Euglena gracilis cDNAsequence (nucleotides 656-663 of SEQ ID NO:23), and since NotI linkerswere used in the original cloning of the Euglena gracilis delta-4desaturase cDNA (see Meyer et al., supra), it is likely that what wascloned was an incomplete, not full-length, transcript for EgDHAsyn2.

The amino acid sequence EgDHAsyn1 (SEQ ID NO:12) was compared toEgDHAsyn2 (SEQ ID NO:22) and EgC20elo1 (SEQ ID NO:6) using the Clustal Wmethod as described above, and the alignment is shown in FIGS. 3A and3B. Compared to EgDHAsyn1 and EgDHAsyn2, EgC20elo1 has a deletion of 7amino acids (i.e., A L D LA [V/I] L) and 2 other amino acidsubstitutions (i.e., W47R, T48I; based on numbering for EgDHAsyn1) atthe N-terminus. After amino acid 289 of EgC20elo1, the sequences arevery different when compared to the DHA synthases. EgDHAsyn1 andEgDHAsyn2 have an additional 498 amino acids at their C-terminal endswith homology to delta-4 fatty acid desaturases, while EgC20elo1 endsafter only 9 additional amino acids. The amino acid sequences ofEgDHAsyn1 (SEQ ID NO:12) and EgDHAsyn2 (SEQ ID NO:22) have 8 amino aciddifferences between the 2 sequences (i.e., V25I, G54V, A305T, L310P,V380I, S491 N, I744T, R747P; based on numbering for EgDHAsyn1). The lastfour differences occur in the delta-4 desaturase domain.

FIGS. 4A and 4B show the Clustal W alignment of the N-terminus ofEgDHAsyn1 (SEQ ID NO:12) and the N-terminus of EgDHAsyn2 (SEQ ID NO:22)with EgC20elo1 (SEQ ID NO:6), Pavlova sp. CCMP459 C20-PUFA Elo (SEQ IDNO:2), Ostreococcus tauri PUFA elongase 2 (SEQ ID NO:25) (NCBI AccessionNo. AAV67798 (GI 55852396), locus AAV67798, CDS AY591336; Meyer et al.,J. Lipid Res. 45(10):1899-1909 (2004)), and Thalassiosira pseudonanaPUFA elongase 2 (SEQ ID NO:26) (NCBI Accession No. AAV67800 (GI55852441), locus AAV67800, CDS AY591338; Meyer et al., J. Lipid Res.,supra). In FIGS. 4A and 4B, the Pavlova, Ostreococcus, and Thalassiosiraproteins are labeled as PavC20elo, OtPUFAelo2, and TpPUFAelo2,respectively.

FIGS. 5A, 5B, 5C, and 5D show the Clustal W alignment of the C-terminusof EgDHAsyn1 (EgDHAsyn1_CT; amino acids 253-793 of SEQ ID NO:12; theN-terminus of EgDHAsyn1 is not shown and is indicated by “ . . . ”) andthe C-terminus of EgDHAsyn2 (EgDHAsyn2_CT; amino acids 253-793 of SEQ IDNO:22, the N-terminus of EgDHAsyn2 is not shown and is indicated by “.”)with Euglena gracilis delta-4 fatty acid desaturase (SEQ ID NO:13),Thraustochytrium aureum delta-4 desaturase (SEQ ID NO:27) (NCBIAccession No. AAN75707(GI 25956288), locus AAN75707, CDS AF391543),Schizochytrium aggregatum delta-4 desaturase (SEQ ID NO:28) (PCTPublication No. WO 2002/090493), Thalassiosira pseudonana delta-4desaturase (SEQ ID NO:29) (NCBI Accession No. AAX14506 (GI 60173017),locus AAX14506, CDS AY817156; Tonon et al., FEBS J. 272 (13):3401-3412(2005)), and Isochrysis galbana delta-4 desaturase (SEQ ID NO:30) (NCBIAccession No. AAV33631 (GI 54307110), locus AAV33631, CDS AY630574;Pereira et al., Biochem. J. 384(2),:357-366 (2004) and PCT PublicationNo. WO 2002/090493). In FIGS. 5A, 5B, 5C, and 5D, the Euglena,Thraustochytrium, Thalassiosira, and Isochrysis proteins are labeled asEgD4, TaD4, TpD4, and IgD4, respectively.

FIG. 6 shows an alignment of interior fragments of EgDHAsyn1 (labeled as“EgDHAsyn1_NCT.pro”; amino acids 253-365 of SEQ ID NO:12) and EgDHAsyn2(labeled as “EgDHAsyn2_NCT.pro”; amino acids 253-365 of SEQ ID NO:22),spanning both the C20 elongase region and the delta-4 desaturase domain(based on homology), with the C-termini of C20 elongases(EgC20elo1_CT.pro, amino acids 246-298 of SEQ ID NO:6; PavC20elo_CT.pro,amino acids 240-277 of SEQ ID NO:2; OtPUFAelo2_CT.pro, amino acids256-300 of SEQ ID NO:25; TpPUFAelo2_CT.pro, amino acids 279-358 of SEQID NO:26) and the N-termini of delta-4 desaturases (EgD4_NT.pro, aminoacids 1-116 of SEQ ID NO:13; TaD4_NT.pro, amino acids 1-47 of SEQ IDNO:27; SaD4_NT.pro, amino acids 1-47 of SEQ ID NO:28; TpD4_NT.pro, aminoacids 1-82 of SEQ ID NO:29; IgD4_NT.pro, amino acids 1-43 of SEQ IDNO:30) is shown. A conserved motif at the C-terminus of all the C20elongase domains (i.e., VLFXXFYXXXY (SEQ ID NO:180)) is also present atthe N-terminus of EgD4 and further supports EgD4 being an incomplete DHAsynthase.

At the C-terminus of the C20 elongase domain for each of EgDHAsyn1,EgDHAsyn2, and EgC20elo1, there is a repeated sequence containing an NGmotif (i.e., KNGK (SEQ ID NO:186), PENGA (SEQ ID NO:187), PENGA (SEQ IDNO:187), and PCENGTV (SEQ ID NO:191); called NG repeats and indicated inFIG. 6 with lines under the sequence). Although the pattern occurs witha high probability of occurrence, a scan of the NG repeated region usingProsite shows the last NG motif (i.e., NGTV) in this region as apotential N-glycosylation site. After the NG repeat region, bothEgDHAsyn1 and EgDHAsyn2 contain a proline-rich region (labeled“Proline-rich linker” in FIG. 6), which may act as a linker between theC20 elongase and delta-4 desaturase domains. The linker may play a rolein keeping the C20 elongase and delta-4 desaturase domains in the properstructural orientation to allow efficient conversion of EPA to DHA.Although the proline-rich linker is shown in FIG. 6 as extending fromP304 to V321 (based on numbering for EgDHAsyn1), the NG repeat region isalso somewhat proline-rich and may also play a role in this linkerfunction.

The nucleotide and corresponding amino acid sequences for theproline-rich linker of EgDHAsyn1, as defined in FIG. 6, are set forth inSEQ ID NO:197 and SEQ ID NO:198, respectively. The nucleotide andcorresponding amino acid sequences for the proline-rich linker ofEgDHAsyn2, as defined in FIG. 6, are set forth in SEQ ID NO:199 and SEQID NO:200, respectively.

The nucleotide and corresponding amino acid sequences for the EgDHAsyn1C20 elongase domain from EgDHAsyn1 are set forth in SEQ ID NO:201 andSEQ ID NO:202, respectively. The nucleotide and corresponding amino acidsequences for the EgDHAsyn2 C20 elongase domain are set forth in SEQ IDNO:203 and SEQ ID NO:204, respectively.

Example 7 Construction of pDMW263

Plasmid pY5-30 (which was previously described in U.S. Pat. No.7,259,255 (the contents of which are hereby incorporated by reference)),is a shuttle plasmid that can replicate both in E. coli and Yarrowialipolytica. Plasmid pY5-30 contains the following: a Yarrowia autonomousreplication sequence (ARS18); a ColE1 plasmid origin of replication; anampicillin-resistance gene (Amp^(R)), for selection in E. coli; aYarrowia LEU2 gene, for selection in Yarrowia; and a chimericTEF::GUS::XPR gene. Plasmid pDMW263 (SEQ ID NO:31) was created frompY5-30, by replacing the TEF promoter with the Yarrowia lipolyticaFBAINm promoter (U.S. Pat. No. 7,202,356), using techniques well knownto one skilled in the art. Briefly, the FBAIN promoter is located in the5′ upstream untranslated region in front of the ‘ATG’ translationinitiation codon of the fructose-bisphosphate aldolase enzyme (E.G.4.1.2.13), encoded by the fba1 gene. This promoter is necessary forexpression and includes a portion of 5′ coding region that has anintron. The modified promoter, FBAINm, has a 52 by deletion between theATG translation initiation codon and the intron of the FBAIN promoter(thereby including only 22 amino acids of the N-terminus) and a newtranslation consensus motif after the intron. Table 20 summarizes thecomponents of pDMW263 (SEQ ID NO:31; also described in PCT PublicationNo. WO 2007/061845).

TABLE 20 Components of Plasmid pDMW263 RE Sites and Nucleotides WithinSEQ ID Description of Fragment and Chimeric Gene NO: 31 Components4992-4296 ARS18 sequence (GenBank Accession No. A17608) SalI/SacIIFBAINm::GUS::XPR, comprising: (8505-2014) FBAINm: FBAINm promoter (PCTPublication No. WO 2005/049805; U.S. Pat. No. 7,202,356) GUS: E. coligene encoding β-glucuronidase (Jefferson, R. A. Nature. 14: 342: 837-838(1989) XPR: ~100 bp of the 3′ region of the Yarrowia Xpr gene (GenBankAccession No. M17741) 6303-8505 Yarrowia Leu2 gene (GenBank AccessionNo. AF260230)

Example 8 Construction of Yarrowia lipolytica Expression Vector pY115and Gateway® Destination Vectors pBY1 and pY159

The NcoI/SalI DNA fragment from pDMW263 (SEQ ID NO:31) (see constructionin Example 7), containing the Yarrowia lipolytica FBAINm promoter, wascloned into the NcoI/SalI DNA fragment of pDMW237 (SEQ ID NO:32),previously described in PCT Publication No. WO 2006/012325 (the contentsof which are hereby incorporated by reference). pDMW237contains asynthetic delta-9 elongase gene derived from Isochrysis galbana andcodon-optimized for expression in Yarrowia lipolytica (IgD9e). In thisway, plasmid pY115 (SEQ ID NO:33; FIG. 7A) was produced. In FIG. 7A, themodified FBAINm promoter is called FBA1+Intron. The modified FBAINmpromoter is referred to in other figures as either FBA1+Intron or YARFBA1 PRO+Intron; these terms are used interchangeably with FBAINm.

Plasmid pY115 (SEQ ID NO:33) was digested with NcoI/NotI, and theresulting DNA ends were filled using Klenow. After filling to form bluntends, the DNA fragments were treated with calf intestinal alkalinephosphatase and separated using agarose gel electrophoresis. The 6989 byfragment containing the Yarrowia lipolytica FBAINm promoter was excisedfrom the agarose gel and purified using the QIAquick® Gel Extraction Kit(Qiagen Inc., Valencia, Calif.), following the manufacturer's protocol.The purified 6989 by fragment was ligated with cassette rfA using theGateway Vector Conversion System (Cat. No. 11823-029, InvitrogenCorporation), following the manufacturer's protocol, to form Yarrowialipolytica Gateway® destination vector pBY1 (SEQ ID NO:34; FIG. 7B).

In constructing pBY1, the filled NcoI site provides an ATG start fortranslation initiation. Thus, genes transferred to this expressionvector are expressed as fusion proteins and must be in the correct frameafter Gateway® cloning. Also, 5′ untranslated sequence results inadditional amino acids being added to the N-terminus of the resultingprotein. For this reason, a second Gateway® destination vector was madewhich had the vector-derived ATG start codon removed, thus allowing fortranslational start from the gene inserted.

The FBAINm promoter was amplified from plasmid pY115 (SEQ ID NO:33),using PCR with oligonucleotide primers oYFBA1 (SEQ ID NO:35) andoYFBA1-6 (SEQ ID NO:36). Primer oYFBA1 (SEQ ID NO:35) was designed tointroduce a BglII site at the 5′ end of the promoter, and primeroYFBA1-6 (SEQ ID NO:36) was designed to introduce a NotI site at the 3′end of the promoter while removing the NcoI site and thus, the ATG startcodon. The resulting PCR fragment was digested with BglII and NotI andcloned into the BglII/NotI fragment of pY115, containing the vectorbackbone, to form pY158 (SEQ ID NO:37).

Plasmid pY158 (SEQ ID NO:37) was digested with NotI, and the resultingDNA ends were filled. After filling to form blunt ends, the DNAfragments were treated with calf intestinal alkaline phosphatase andseparated using agarose gel electrophoresis. The 6992 by fragmentcontaining the Yarrowia lipolytica FBAINm promoter was excised from theagarose gel and purified using the QIAquick® Gel Extraction Kit (QiagenInc., Valencia, Calif.), following the manufacturer's protocol. Thepurified 6992 by fragment was ligated with cassette rfA using theGateway Vector Conversion System (Cat. No. 11823-029, InvitrogenCorporation), following the manufacturer's protocol, to form Yarrowialipolytica Gateway® destination vector pY159 (SEQ ID NO:38; FIG. 7C).

Example 9 Construction of Yarrowia lipolytica Expression VectorspBY-EqC20elo1 (EgC20elo1), pY132 (EgDHAsyn1), pY161 (EgDHAsyn1) andpY164 (EgDHAsyn2)

Plasmid was purified from clones eeg1c.pk005.p14.f (Example 3),eeg1c.pk016.e6.f (Example 4), and eeg1c-1 (Example 5) using the QIAprep®Spin Miniprep Kit (Qiagen Inc., Valencia, Calif.), following themanufacturer's protocol. Using the Gateway® LR Clonase™ II enzyme mix(Cat. No. 11791-020, Invitrogen Corporation) and following themanufacturer's protocol, the cDNA inserts from eeg1c.pk001.p14.f(comprising EgC20elo1) and eeg1c.pk016.e6.f (comprising EgDHAsyn1) weretransferred to pBY1 (SEQ ID NO:34; FIG. 7B) to form pBY-EgC20elo1 (SEQID NO:39, FIG. 7D) and pY132 (SEQ ID NO:40; FIG. 8A), respectively. ThecDNA insert from eeg1c-1 (comprising EgDHAsyn2) was not transferred topBY1, because it would have resulted in the wrong translation framebeing expressed.

Using the Gateway® LR Clonase™ II enzyme mix (Cat. No. 11791-020,Invitrogen Corporation) and following the manufacturer's protocol, thecDNA inserts from eeg1c.pk016.e6.f and eeg1c-1 were transferred to pY159(SEQ ID NO:38; Example 8) to form pY161 (SEQ ID NO:41, FIG. 8B) andpY164 (SEQ ID NO:42; FIG. 8C), respectively.

Example 10 Construction of Yarrowia lipolytica Expression Vectors pY141(EgDHAsyn1*), pY143 (EgDHAsyn1*C20EloDom1) and pY149(EgDHAsyn1*C20EloDom2Linker)

EgDHAsyn1 was amplified from clone eeg1c.pk001.e6.f with oligonucleotideprimers EgEPAEloDom-5 (SEQ ID NO:43) and oEUG el4-3 (SEQ ID NO:44),using the Phusion™ High-Fidelity DNA Polymerase (Cat. No. F553S,Finnzymes Oy, Finland) following the manufacturer's protocol. Theresulting DNA fragment was cloned into the pCR-Blunt® cloning vectorusing the Zero Blunt® PCR Cloning Kit (Invitrogen Corporation),following the manufacturer's protocol, to produce pKR1062 (SEQ IDNO:45).

An internal NcoI site at nucleotides 619-624 was removed from EgDHAsyn1in pKR1062 using the Quickchange® Site Directed Mutagenesis kit (Cat.No. 200518, Stratagene, La Jolla, Calif.), with oligonucleotidesEgEloD4Mut-5 (SEQ ID NO:46) and EgEloD4Mut-3 (SEQ ID NO:47), followingthe manufacturer's protocol. After extensive sequencing, a clone withthe NcoI site removed (i.e., a ccatgg to ccttgg mutation) and no furthernucleotide changes made was chosen for further study. This clone wasdesignated pLF115-7 (SEQ ID NO:48). The nucleotide sequence forEgDHAsyn1 having the NcoI site removed (EgDHAsyn1*) is set forth in SEQID NO:205. The corresponding amino acid sequence is identical to SEQ IDNO:12.

Construction Of Plasmid pY141, Expressing EgDHAsyn1*:

The NcoI/NotI DNA fragment from pLF115-7 (SEQ ID NO:48), containingEgDHAsyn1 (SEQ ID NO:205; without the internal NcoI site; at nt 621 ofthe EgDHAsyn1 CDS; ccatgg to ccttgg), was cloned into the NcoI/NotI DNAfragment from pY115, containing the Yarrowia lipolytica FBAINm promoter,to produce pY141 (SEQ ID NO:49; FIG. 8D). Thus. plasmid pY141 containsthe full length EgDHAsyn1* gene (labeled as “EgDHAsyn1(-NcoI)” in FIG.),under control of the Yarrowia lipolytica FBAINm promoter (PCTPublication No. WO 2005/049805; U.S. Pat. No. 7,202,356; labeled as“Fba1+Intron” in FIG.), and the Pex20 terminator sequence from YarrowiaPex20 gene (GenBank Accession No. AF054613).

Construction Of Plasmid pY143, Expressing EgDHAsyn1-C20EloDom1:

The nucleotide sequence for the EgDHAsyn1* C20 elongase domain(EgDHAsyn1C20EloDom1) in pY141 is set forth in SEQ ID NO:206 (identicalto SEQ ID NO:201 but NcoI site removed). The corresponding amino acidsequence is identical to SEQ ID NO:202.

The EgDHAsyn1C20EloDom1 (SEQ ID NO:206) was amplified from pLF115-7 witholigonucleotide primers EgEPAEloDom-5 (SEQ ID NO:43) and EgDPAEloDom-3(SEQ ID NO:50) using the Phusion™ High-Fidelity DNA Polymerase (Cat. No.F553S, Finnzymes Oy, Finland) following the manufacturer's protocol. Theresulting DNA fragment was cloned into the pCR-Blunt® cloning vectorusing the Zero Blunt® PCR Cloning Kit (Invitrogen Corporation),following the manufacturer's protocol, to produce pHD16 (SEQ ID NO:51).

The NcoI/NotI DNA fragment from pHD16 (SEQ ID NO:51), containing theEgDHAsyn1C20EloDom1 (without the internal NcoI site), was cloned intothe NcoI/NotI DNA fragment from pY115, containing the Yarrowialipolytica FBAINm promoter, to produce pY143 (SEQ ID NO:52; FIG. 9A).Plasmid pY143 contains the N-terminal domain of EgDHAsyn1*(EgDHAsyn1C20EloDom1) and does not include the proline-rich linker ordelta-4 desaturase domain.

Construction of Plasmid pY149, Expressing EgDHAsyn1-C20EloDom2Linker:

The EgDHAsyn1* C20 elongase domain (SEQ ID NO:206) and proline-richlinker (SEQ ID NO:197), were amplified from pLF115-7 (SEQ ID NO:48) witholigonucleotide primers EgEPAEloDom-5 (SEQ ID NO:43) and oEUGsyn6-2 (SEQID NO:53) using the Phusion™ High-Fidelity DNA Polymerase (Cat. No.F553S, Finnzymes Oy, Finland) following the manufacturer's protocol. Theresulting DNA fragment was cloned into the pCR-Blunt® cloning vectorusing the Zero Blunt® PCR Cloning Kit (Invitrogen Corporation),following the manufacturer's protocol, to produce pKR1071 (SEQ IDNO:54).

The NcoI/Ecl13211 DNA fragment from pKR1071 (SEQ ID NO:54) was clonedinto the NcoI/NotI DNA fragment from pY115 (where the NotI site had beenfilled in), containing the Yarrowia lipolytica FBAINm promoter, toproduce pY149 (SEQ ID NO:55; FIG. 9B). Plasmid pY149 contains theEgDHAsyn1C20EloDom1/proline-rich linker fusion gene (i.e.,EgDHAsyn1C20EloDom2Linker; SEQ ID NO:207), but does not contain thedelta-4 desaturase domain. The amino acid sequence ofEgDHAsyn1C20EloDom2Linker is set forth in SEQ ID NO:208. In addition tothe amino acids from EgDHAsyn1* C20 elongase domain and proline-richlinker, an additional 4 amino acids (i.e., SCRT) were added after thelinker region as a result of how the fragment was synthesized andcloned.

Example 11 Construction of Yarrowia lipolytica Expression Vectors forGeneration of Novel C20 Elongase/Delta-4 Desaturase Fusion Proteins

In order to synthesize novel C20 elongase/delta-4 desaturase fusionproteins, a unique SbfI site was added to the 3′ end of the C20 elongasedomain of EgDHAsyn1* after the proline-rich linker region(EgDHAsyn1C20EloDom3Linker). EgDHAsyn1C20EloDom3 was amplified frompLF115-7 (SEQ ID NO:48) with oligonucleotide primers EgEPAEloDom-5 (SEQID NO:43) and oEUGsyn6-3 (SEQ ID NO:56) using the Phusion™ High-FidelityDNA Polymerase (Cat. No. F553S, Finnzymes Oy, Finland) following themanufacturer's protocol. The resulting DNA fragment was cloned into thepCR-Blunt® cloning vector using the Zero Blunt® PCR Cloning Kit(Invitrogen Corporation), following the manufacturer's protocol, toproduce pKR1091 (SEQ ID NO:57).

The NcoI/Ecl13611 DNA fragment from pKR1091 (SEQ ID NO:57), containingEgDHAsyn1C20EloDom3Linker, was cloned into the NcoI/NotI DNA fragmentfrom pY115 (where the NotI was filled to form a blunt end), containingthe Yarrowia lipolytica FBAINm promoter, to produce pY155 (SEQ IDNO:58).

In order to synthesize novel C20 elongase/delta-4 desaturase fusionproteins, a unique SbfI site was added to the 5′ end of various delta-4desaturases. In each case, the SbfI site is located after the ATG startsite of each coding sequence and resulted in the addition and/orreplacement of a few amino acids at the N-terminus of the delta-4desaturase coded for by the genes.

Construction Of Plasmid pY156, Expressing EgDHAsyn1-C20EloDom3-IgD4*:

The Isochrysis galbana delta-4 desaturase (SEQ ID NO:209; IgD4) wasamplified from pRIG6 (previously described in PCT Publication No. WO2002/090493) with oligonucleotides ORIG6-1 (SEQ ID NO:59) and ORIG6-2(SEQ ID NO:60) using the Phusion™ High-Fidelity DNA Polymerase (Cat. No.F553S) following the manufacturer's protocol. The resulting DNAfragment, which contains the IgD4 CDS and is identical to SEQ ID NO:209except that an SbfI site was added at the 5′ end after the start codon(IgD4*; SEQ ID NO:210), was cloned into the pCR-Blunt® cloning vectorusing the Zero Blunt® PCR Cloning Kit (Invitrogen Corporation),following the manufacturer's protocol, to produce pKR1067 (SEQ IDNO:61). The amino acid sequence for IgD4* from pKR1067 is set forth inSEQ ID NO:211 and is identical to that to IgD4 (SEQ ID NO:30) exceptthat the first 4 amino acids (i.e., MCNA) have been changed to MALQ dueto the addition of the SbfI site in the nucleotide sequence.

The NcoI/NotI DNA fragment from pKR1067 (SEQ ID NO:61), containingIgD4*, was cloned into the NcoI/NotI DNA fragment from pY115, containingthe Yarrowia lipolytica FBAINm promoter, to produce pY150 (SEQ ID NO:62;FIG. 9C). In FIG. 9C, IgD4* is labeled as “Ig d4 DS”. In this way, IgD4*could be expressed alone in Yarrowia.

The XbaI/SbfI DNA fragment from pKR1091 (SEQ ID NO:57; supra),containing EgDHAsyn1C20EloDom3Linker, was cloned into the XbaI/SbfI DNAfragment from pKR1067 (SEQ ID NO:61), containing IgD4*, to producepKR1097 (SEQ ID NO:63). Thus, an in-frame fusion was made betweenEgDHAsyn1C20EloDom3Linker and IgD4*, separated by the proline-richlinker region (called EgDHAsyn1C20EloDom3-IgD4; SEQ ID NO:212). Theamino acid sequence for EgDHAsyn1C20EloDom3-1gD4 is set forth in SEQ IDNO:213.

The NcoI/NotI DNA fragment from pKR1097 (SEQ ID NO:63), containing theEgDHAsyn1C20EloDom3-1gD4, was cloned into the NcoI/NotI DNA fragmentfrom pY115, containing the Yarrowia lipolytica FBAINm promoter, toproduce pY156 (SEQ ID NO:64; FIG. 9D). In FIG. 9D, theEgDHAsyn1C20EloDom3-IgD4 is labeled as “EGel-IGd4”.

Construction Of Plasmid pY152, Expressing EgDHAsyn1-D4Dom1*:

A region of the C-terminus of EgDHAsyn1* (SEQ ID NO:205) containing thedelta-4 desaturase domain (EgDHAsyn1D4Dom1; SEQ ID NO:214; correspondingamino acid sequence for EgDHAsyn1D4Dom1 is set forth in SEQ ID NO:215),starting just after the end of the proline-rich linker region, wasamplified from pLF115-7 (as described in Example 10) witholigonucleotides oEGslne6-1 (SEQ ID NO:65) and oEUGel4-3 (SEQ ID NO:44)using the Phusion™ High-Fidelity DNA Polymerase (Cat. No. F553S)following the manufacturer's protocol. Oligonucleotide oEGslne6-1 (SEQID NO:65) introduced an ATG start codon at the 5′ end of the PCR productfollowed by an SbfI site. The resulting DNA fragment was cloned into thepCR-Blunt® cloning vector using the Zero Blunt® PCR Cloning Kit(Invitrogen Corporation), following the manufacturer's protocol, toproduce pKR1069 (SEQ ID NO:66). The new CDS and amino acid sequencescontaining EgDHAsyn1D4Dom1 from pKR1069 (i.e., EgDHAsyn1D4Dom1*) are setforth in SEQ ID NO:216 and SEQ ID NO:217, respectively. The amino acidsequence for EgDHAsyn1D4Dom1* (SEQ ID NO:217) is identical to that ofEgDHAsyn1D4Dom1 (SEQ ID NO:215), except that the first 2 amino acids(i.e., SG) have been changed to MAL due to the addition of the SbfI sitein the nucleotide sequence.

The NcoI/NotI DNA fragment from pKR1069 (SEQ ID NO:66), containing theEgDHAsyn1D4Dom1*, was cloned into the NcoI/NotI DNA fragment from pY115,containing the Yarrowia lipolytica FBAINm promoter, to produce pY152(SEQ ID NO:67; FIG. 10A). In FIG. 10A, the EgDHAsyn1D4Dom1* is labeledas “EUG d4 (fus test)”. In this way, the EgDHAsyn1D4Dom1* could beexpressed alone in Yarrowia.

Construction Of Plasmid pY157, Expressing EgDHAsyn1-C20EloDom3-EgD4Dom1:

The XbaI/SbfI DNA fragment from pKR1091 (SEQ ID NO:57), containingEgDHAsyn1C20EloDom3-Linker, was cloned into the XbaI/SbfI DNA fragmentfrom pKR1069, containing the EgDHAsyn1D4Dom1*, to produce pKR1099 (SEQID NO:68). In this way, an in-frame fusion was made between theEgDHAsyn1C20EloDom and the EgDHAsyn1D4Dom1*, separated by theproline-rich linker region (called EgDHAsyn1C20EloDom3-EgD4Dom1; SEQ IDNO:218). The amino acid sequence of EgDHAsyn1C20EloDom3-EgD4Dom1 (SEQ IDNO:219) is almost identical to EgDHAsyn1 except one amino acid (i.e.,G323L based on numbering for EgDHAsyn1) was changed due to the SbfIcloning site and fusion junction.

The NcoI/NotI DNA fragment from pKR1099 (SEQ ID NO:68), containing theEgDHAsyn1C20EloDom3-EgD4Dom1, was cloned into the NcoI/NotI DNA fragmentfrom pY115, containing the Yarrowia lipolytica FBAINm promoter, toproduce pY157 (SEQ ID NO:69; FIG. 10B). In FIG. 10B, theEgDHAsyn1C20EloDom3-EgD4Dom1 is labeled as “EGel-EGd4 fus”.

Construction Of Plasmid pY153, Expressing EgDHAsyn1*D4Dom2:

A region of the C-terminus of EgDHAsyn1 containing the delta-4desaturase domain and some of the C20 elongase domain (EgDHAsyn1D4Dom2;SEQ ID NO:220; corresponding amino acid sequence for EgDHAsyn1D4Dom2 isset forth in SEQ ID NO:221), which corresponds to the amino acidsequence identified as EgD4 (SEQ ID NO:13; Meyer et al., Biochemistry42(32):9779-9788 (2003)), was amplified from pLF115-7 (described inExample 10) with oligonucleotides oEUGel4-4 (SEQ ID NO:70) and oEUGel4-3(SEQ ID NO:44) using the Phusion™ High-Fidelity DNA Polymerase (Cat. No.F553S) following the manufacturer's protocol. The resulting DNA fragmentwas cloned into the pCR-Blunt® cloning vector using the Zero Blunt®PCRCloning Kit (Invitrogen Corporation), following the manufacturer'sprotocol, to produce pKR1073 (SEQ ID NO:71).

The PciI/NotI DNA fragment from pKR1073 (SEQ ID NO:71), containing theEgDHAsyn1D4Dom2, was cloned into the NcoI/NotI DNA fragment from pY115,containing the Yarrowia lipolytica FBAINm promoter, to produce pY153(SEQ ID NO:72; FIG. 10C). In FIG. 10C, the EgDHAsyn1D4Dom2 is labeled asEUG d4 (HZ). In this way, the EgDHAsynD4Dom2 could be expressed alone inYarrowia.

Construction Of Plasmid pY160, Expressing EgDHAsyn1-C20EloDom3-SaD4*:

The Schizochytrium aggregatum delta-4 desaturase (SEQ ID NO:222; SaD4)was amplified from pRSA-1 (previously described in PCT Publication No.WO 2002/090493) with oligonucleotides oRSA1-1 (SEQ ID NO:73) and oRSA1-2(SEQ ID NO:74) using the Phusion™ High-Fidelity DNA Polymerase (Cat. No.F553S) following the manufacturer's protocol. The resulting DNAfragment, which contains the SaD4 CDS and is identical to SEQ ID NO:222,except that an SbfI site was added at the 5′ end after the start codon(SaD4*; SEQ ID NO:223), was cloned into the pCR-Blunt® cloning vectorusing the Zero Blunt® PCR Cloning Kit (Invitrogen Corporation),following the manufacturer's protocol, to produce pKR1068 (SEQ IDNO:75). The amino sequence for SaD4* from pKR1068 is set forth in SEQ IDNO:224 and is identical to that to SaD4 (SEQ ID NO:28) except that thefirst 3 amino acids (i.e., MTV) have been changed to MALQ due to theaddition of the SbfI site in the nucleotide sequence.

The NcoI/NotI DNA fragment from pKR1068 (SEQ ID NO:75) (partial digestto avoid internal NcoI site), containing the SaD4*, was cloned into theNcoI/NotI DNA fragment from pY115, containing the Yarrowia lipolyticaFBAINm promoter, to produce pY151 (SEQ ID NO:76; FIG. 10D). In FIG. 10D,the SaD4* is labeled as “RSA d4 DS”. In this way, the SaD4* could beexpressed alone in Yarrowia.

The SbfI/NotI DNA fragment from pKR1068 (SEQ ID NO:75), containing theSaD4*, was cloned into the SbfI/NotI DNA fragment from pY157 (SEQ IDNO:69), containing the EgDHAsyn1C20EloDom3Linker, to produce pY160 (SEQID NO:77; FIG. 11). In this way, an in-frame fusion was made between theEgDHAsyn1C20EloDom3 and the SaD4*, separated by the proline-rich linkerregion (i.e., EgDHAsyn1C20EloDom3-SaD4; SEQ ID NO:225). The amino acidsequence for EgDHAsyn1C20EloDom3-SaD4 is set forth in SEQ ID NO:226.

Example 12 Euglena anabaena Growth Conditions, Lipid Profile and mRNAIsolation

Euglena anabaena was obtained from Dr. Richard Triemer's lab at MichiganState University (East Lansing, Mich.). Approximately 2 mL of culturewere removed for lipid analysis and centrifuged at 1,800×g for 5 min.The pellet was washed once with water and re-centrifuged. The resultingpellet was dried for 5 min under vacuum, resuspended in 100 μL oftrimethylsulfonium hydroxide (TMSH), and incubated at room temperaturefor 15 min with shaking. After this, 0.5 mL of hexane were added, andthe vials were incubated for 15 min at room temperature with shaking.Fatty acid methyl esters (5 μL injected from hexane layer) wereseparated and quantified using a Hewlett-Packard 6890 Gas Chromatographfitted with an Omegawax 320 fused silica capillary column (Supelco Inc.,Cat. No. 24152). The oven temperature was programmed to hold at 170° C.for 1.0 min, increase to 240° C. at 5° C./min, and then hold for anadditional 1.0 min. Carrier gas was supplied by a Whatman hydrogengenerator. Retention times were compared to those for methyl esters ofstandards commercially available (Nu-Chek Prep, Inc. Cat. No. U-99-A),and the resulting chromatogram is shown in FIG. 12. The presence of EPAand DHA in the fatty acid profile suggested that Euglena anabaena wouldbe a good source for long-chain PUFA biosynthetic genes such as, but notlimited to, C20 elongases, delta-4 desaturases, and/or DHA synthases.

The remaining 5 mL of an actively growing culture was transferred into25 mL of AF-6 Medium (Watanabe & Hiroki, NIES-Collection List ofStrains, 5^(th) ed., National Institute for Environmental Studies,Tsukuba, 127 pp (2004)) in a 125 mL glass flask. Euglena anabaenacultures were grown at 22° C. with a 16 hr light, 8 hr dark cycle for 2weeks with very gentle agitation.

After 2 weeks, the culture (25 mL) was transferred to 100 mL of AF-6medium in a 500 mL glass bottle, and the culture was grown for 1 monthas described above. After this time, two 50 mL aliquots were transferredinto two separate 500 mL glass bottles containing 250 mL of AF-6 medium,and the cultures were grown for two months as described above (giving atotal of ˜600 mL of culture). After this, the cultures were pelleted bycentrifugation at 1,800×g for 10 min, washed once with water, andre-centrifuged. Total RNA was extracted from one of the resultingpellets using the RNA STAT-60™ reagent (TEL-TEST, Inc., Friendswood,Tex.) and following the manufacturer's protocol (use 5 mL of reagent,dissolved RNA in 0.5 mL of water). In this way, 340 μg of total RNA (680ug/mL) were obtained from the pellet. The remaining pellet was frozen inliquid nitrogen and stored at −80° C. The mRNA was isolated from all 340μg of total RNA using the mRNA Purification Kit (Amersham Biosciences,Piscataway, N.J.), following the manufacturer's protocol. In this way,9.0 μg of mRNA were obtained.

Example 13 Euglena anabaena cDNA Synthesis, Library Construction andIdentification of DHA Synthases from cDNA Library eug1c

A cDNA library was generated using the Cloneminer™ cDNA LibraryConstruction Kit (Cat. No. 18249-029, Invitrogen Corporation, Carlsbad,Calif.), following the manufacturer's protocol (Version B, 25-0608).Using the non-radiolabeling method, cDNA was synthesized from 5.12 μg ofmRNA (Example 12) using the Biotin-attB2-Oligo(dT) primer. Aftersynthesis of the first and second strand, the attB1 adapter was added;ligation was performed; and the cDNA was size fractionated using columnchromatography. DNA from fractions were concentrated, recombined intopDONR™ 222, and transformed into E. coli ElectroMAX™ DH10B™ T1Phage-Resistant cells (Invitrogen Corporation). The Euglena anabaenalibrary was named eug1c.

Approximately 17,000 clones of cDNA library eug1c were plated onto 3large square (24 cm×24 cm) petri plates (Corning, Corning, N.Y.), eachcontaining LB+50 μg/mL kanamycin agar media. Cells were grown,transferred to Biodyne B membrane, and hybridized with a labeledNcoI/NotI DNA fragment, containing EgDHAsyn1*, from pY141, exactly asdescribed in Example 5. In this way, 11 positive clones were identified(designated as eug1c-1 to eug1c-11).

The positive clones were grown, and DNA was purified and sequenced asdescribed in Example 2 using vector-primed M13F Universal primer (SEQ IDNO:1), vector-primed M13-28Rev primer (SEQ ID NO:14), and the poly(A)tail-primed WobbleT oligonucleotides. Based on initial sequence data,additional internal fragment sequence was obtained in a similar wayusing oligonucleotides EaDHAsyn5′ (SEQ ID NO:78), EaDHAsyn5′2 (SEQ IDNO:79), EaDHAsyn5′3 (SEQ ID NO:80), EaDHAsyn5′4 (SEQ ID NO:81),EaDHAsyn3′ (SEQ ID NO:82), EaDHAsyn3′2 (SEQ ID NO:83), EaDHAsyn3′3 (SEQID NO:84), EaDHAsyn3′4 (SEQ ID NO:85), and EaDHAsyn3′S (SEQ ID NO:86).In this way, the full insert sequences of the eug1c clones wereobtained.

Sequences were aligned and compared using Sequencher™ (Version 4.2, GeneCodes Corporation, Ann Arbor, Mich.), and in this way, the clones couldbe categorized into one of four distinct groups based on insert sequence(identified as EaDHAsyn1 to EaDHAsyn4). Representative clones containingthe cDNA for each class of sequence were chosen for further study, andsequences for each representative plasmid (i.e., pLF117-1, pLF117-2,pLF117-3 and pLF117-4) are shown as SEQ ID NO: 87, SEQ ID NO:88, SEQ IDNO:89, and SEQ ID NO:90, respectively. The sequence of pLF117-1 shown bya string of NNNN's represents a region of the polyA tail which was notsequenced. The coding sequences for EaDHAsyn1, EaDHAsyn2, EaDHAsyn3, andEaDHAsyn4 are shown as SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, and SEQID NO:94, respectively. The corresponding amino acid sequences forEaDHAsyn1, EaDHAsyn2, EaDHAsyn3, and EaDHAsyn4 are shown as SEQ IDNO:95, SEQ ID NO:96, SEQ ID NO:97, and SEQ ID NO:98, respectively.

The amino acid sequences for EaDHAsyn1 (SEQ ID NO:95), EaDHAsyn2 (SEQ IDNO:96), EaDHAsyn3 (SEQ ID NO:97), and EaDHAsyn4 (SEQ ID NO:98) wereevaluated by BLASTP as described in Example 3 and, as was the case forEgDHAsyn1 (SEQ ID NO:12) and EgDHAsyn2 (SEQ ID NO:22), all four EaDHAsynsequences were also found to be similar to both C20-PUFA Elo and delta-4fatty acid desaturases. The N-termini of EaDHAsyn1 (SEQ ID NO:95),EaDHAsyn2 (SEQ ID NO:96), EaDHAsyn3 (SEQ ID NO:97), and EaDHAsyn4 (SEQID NO:98) each yielded a pLog value of 58.5 (E value of 3e-59; 114/247identical amino acids; 46% identity) versus the Pavlova sp. CCMP459C20-PUFA Elo (SEQ ID NO:2). The C-termini of EaDHAsyn1 (SEQ ID NO:95),EaDHAsyn2 (SEQ ID NO:96), EaDHAsyn3 (SEQ ID NO:97), and EaDHAsyn4 (SEQID NO:98) yielded E values of 0.0 (378/538 identical amino acids; 70%identity), 0.0 (378/538 identical amino acids; 70% identity), 0.0(379/538 identical amino acids; 70% identity), and 0.0 (368/522identical amino acids; 70% identity), respectively, versus the aminoacid sequence of delta-4 fatty acid desaturase from Euglena gracilis(SEQ ID NO:13). BLAST scores and probabilities indicate that the instantnucleic acid fragments encode entire Euglena anabaena C20-PUFAElo/delta-4 fatty acid desaturases.

The amino acid sequences for EaDHAsyn1 (SEQ ID NO:95), EaDHAsyn2 (SEQ IDNO:96), EaDHAsyn3 (SEQ ID NO:97), and EaDHAsyn4 (SEQ ID NO:98) werecompared using the Clustal W method as described in Example 6, and thealignment is shown in FIGS. 13A, 13B, and 13C. Interestingly, due to asingle by deletion in the nucleotide sequence, the C-terminus of theresulting amino acid sequence for EaDHAsyn4 (approximately last 35 aminoacids) is highly divergent and smaller than the other three EaDHAsynproteins.

When compared to the amino acid sequence of EgDHAsyn1 (SEQ ID NO:12)using BLASTP, the amino acid sequences of EaDHAsyn1 (SEQ ID NO:95),EaDHAsyn2 (SEQ ID NO:96), EaDHAsyn3 (SEQ ID NO:97), and EaDHAsyn4 (SEQID NO:98) were 70% (558/791), 70% (558/791), 70% (559/791) and 70%(548/775) identical, respectively.

As was the case for EgDHAsyn1 (SEQ ID NO:12) and EgDHAsyn2 (SEQ IDNO:22), all four EaDHAsyn sequences have a proline-rich linker region(from approximately P300 to T332 based on numbering for EaDHAsyn1). Thelinker appears to be slightly longer than that for EgDHAsyn1 (SEQ IDNO:12) or EgDHAsyn2 (SEQ ID NO:22). All four EaDHAsyn sequences alsolack the NG repeat motif found upstream of the proline-rich motif ofEgDHAsyn1 and EgDHAsyn2; but, this region, as was the case for EgDHAsyn1and EgDHAsyn2, is also slightly proline-rich in all four EaDHAsynsequences and may play a role in the linker function.

The nucleotide sequences for the C20 elongase domains of EaDHAsyn1,EaDHAsyn2, EaDHAsyn3, and EaDHAsyn4 are set forth in SEQ ID NO:227, SEQID NO:228, SEQ ID NO:229, and SEQ ID NO:230, respectively. The aminoacid sequences for the C20 elongase domains of EaDHAsyn1, EaDHAsyn2, andEaDHAsyn3 are set forth in SEQ ID NO:231, SEQ ID NO:232, and SEQ IDNO:233, respectively. The amino acid sequence of the C20 elongase domainof EaDHAsyn4 is identical to that for EaDHAsyn1.

The nucleotide and amino acid sequences for the proline-rich linker ofEaDHAsyn1 are set forth in SEQ ID NO:234 and SEQ ID NO:235,respectively. The nucleotide and amino acid sequences for theproline-rich linkers of EaDHAsyn2, EaDHAsyn3, and EaDHAsyn4 areidentical to that for EaDHAsyn1.

The nucleotide sequences for the delta-4 desaturase domain 1 of each ofEaDHAsyn1, EaDHAsyn2, and EaDHAsyn4 are set forth in SEQ ID NO:236, SEQID NO:237, and SEQ ID NO:238, respectively. The amino acid sequences forthe delta-4 desaturase domains of EaDHAsyn1, EaDHAsyn2, and EaDHAsyn4are set forth in SEQ ID NO:239, SEQ ID NO:240, and SEQ ID NO:241,respectively. The nucleotide and amino acid sequence of the delta-4desaturase domain 1 of EaDHAsyn3 is identical to that of EaDHAsyn1.

The nucleotide sequences for the delta-4 desaturase domain 2 ofEaDHAsyn1, EaDHAsyn2, EaDHAsyn3, and EaDHAsyn4, including theproline-rich linker and a portion of the 3′ end of the C20 elongasedomain, are set forth in SEQ ID NO:242, SEQ ID NO:243, SEQ ID NO:244,and SEQ ID NO:245, respectively. The amino acid sequences for thedelta-4 desaturase domains of EaDHAsyn1, EaDHAsyn2, EaDHAsyn3, andEaDHAsyn4 are set forth in SEQ ID NO:246, SEQ ID NO:247, SEQ ID NO:248,and SEQ ID NO:249, respectively.

FIG. 29 summarizes the Euglena anabaena DHA synthase domain sequences.

Example 14 Construction of Yarrowia lipolytica Expression Vectors pY165,pY166, pY167 and pY168

Using the Gateway® LR Clonase™ II enzyme mix (Cat. No. 11791-020,Invitrogen Corporation) and following the manufacturer's protocol, thecDNA inserts from pLF117-1 (SEQ ID NO:87), pLF117-2 (SEQ ID NO:88),pLF117-3 (SEQ ID NO:89), and pLF117-4 (SEQ ID NO:90) were transferred topY159 (SEQ ID NO:38; Example 8) to form pY165 (SEQ ID NO:99, FIG. 14A),pY166 (SEQ ID NO:100; FIG. 14B), pY167 (SEQ ID NO:101; FIG. 14C), andpY168 (SEQ ID NO:102; FIG. 14D), respectively. Thus, each plasmidcontains the full length EaDHAsyn gene, under control of the Yarrowialipolytica FBAINm promoter (PCT Publication No. WO 2005/049805; U.S.Pat. No. 7,202,356; labeled as “Yar Fba1 Pro+Intron” in FIG.), and thePex20 terminator sequence from Yarrowia Pex20 gene (GenBank AccessionNo. AF054613).

Example 15 Construction of Soybean Expression Vector pKR1061ForCo-Expression of the Euglena gracilis DHA Synthase 1 (EgDHAsyn1) Withthe Saprolegnia diclina Delta-17 Desaturase (SdD17)

The present Example describes construction of a soybean vector forco-expression of EgDHAsyn1 (SEQ ID NO:12) with SdD17 and a hygromycinphosphotransferase selectable marker (hpt).

EgDHAsyn1 was amplified from pKR1049 (clone eeg1c.pk016.e6.f) witholigonucleotide primers oEGel2-1 (SEQ ID NO:103) and oEUG el4-3 (SEQ IDNO:44), using the Phusion™ High-Fidelity DNA Polymerase (Cat. No. F553S,Finnzymes Oy, Finland) following the manufacturer's protocol. Theresulting DNA fragment was cloned into the pCR-Blunt® cloning vectorusing the Zero Blunt® PCR Cloning Kit (Invitrogen Corporation),following the manufacturer's protocol, to produce pKR1055 (SEQ IDNO:104).

A starting plasmid pKR72 (ATCC Accession No. PTA-6019; SEQ ID NO:105,7085 by sequence), a derivative of pKS123 which was previously describedin PCT Publication No. WO 2002/008269 (the contents of which are herebyincorporated by reference), contains the hygromycin B phosphotransferasegene (HPT) (Gritz, L. and Davies, J., Gene 25:179-188 (1983)), flankedby the T7 promoter and transcription terminator (T7prom/HPT/T7termcassette), and a bacterial origin of replication (ori) for selection andreplication in bacteria (e.g., E. coli). In addition, pKR72 alsocontains HPT, flanked by the 35S promoter (Odell et al., Nature313:810-812 (1985)) and NOS3′ transcription terminator (Depicker et al.,J. Mol. Appl. Genet. 1:561-570 (1982)) (35S/HPT/NOS3′ cassette), forselection in plants such as soybean. pKR72 also contains a NotIrestriction site, flanked by the promoter for the α′ subunit ofβ-conglycinin (Beachy et al., EMBO J. 4:3047-3053 (1985)) and the 3′transcription termination region of the phaseolin gene (Doyle et al., J.Biol. Chem. 261:9228-9238 (1986)), thus allowing for strongtissue-specific expression in the seeds of soybean of genes cloned intothe NotI site.

The βcon/NotI/Phas3′ cassette in plasmid pKR72 (SEQ ID NO:105, havingATCC Accession No. PTA-6019) was amplified using oligonucleotide primersoCon-1 (SEQ ID NO:106) and oCon-2 (SEQ ID NO:107) using the VentR® DNAPolymerase (Catalog No. M0254S, New England Biolabs Inc., Beverly,Mass.) following the manufacturer's protocol. The resulting DNA fragmentwas digested with XbaI and cloned into the XbaI site of pUC19, toproduce pKR179 (SEQ ID NO:108).

EgDHAsyn1 was released from pKR1055 (SEQ ID NO:104) by digestion withNotI and was cloned into the NotI site of plasmid pKR179 (SEQ ID NO:108)to produce pKR1057 (SEQ ID NO:109).

The SbfI fragment of pKR1057 (SEQ ID NO:109), containing theβcon/EgDHAsyn1/Phas3′ cassette was cloned into the SbfI site of pKR328(SEQ ID NO:110; which is described in PCT Publication No. WO 2004/071467and the contents of which are hereby incorporated by reference),containing SdD17, to produce vector pKR1061 (SEQ ID NO:111). A schematicdepiction of pKR1061 is shown in FIG. 15A.

Example 16 Construction of Soybean Expression Vector pKR973ForCo-Expression of the Pavlova lutheri Delta-8 Desaturase (PavD8) with theEuglena gracilis Delta-9 Elongase (EgD9elo) and the Mortierella alpinaDelta-5 Desaturase (MaD5)

Euglena gracilis Delta-9 Elongase (EgD9elo):

A clone from the Euglena cDNA library (eeg1c), called eeg1c.pk001.n5f,containing the Euglena gracilis delta-9 elongase (EgD9elo; SEQ IDNO:112; which is described in U.S. application Ser. No. 11/601,563(filed Nov. 16, 2006, which published May 24, 2007 asUS-2007-0118929-A1; Attorney Docket No. BB-1562) the contents of whichare hereby incorporated by reference) was used as template to amplifyEgD9elo with oligonucleotide primers oEugEL1-1 (SEQ ID NO:113) andoEugEL1-2 (SEQ ID NO:114) using the VentR® DNA Polymerase (Cat. No.M0254S, New England Biolabs Inc., Beverly, Mass.) following themanufacturer's protocol. The resulting DNA fragment was cloned into thepCR-Blunt® cloning vector using the Zero Blunt® PCR Cloning Kit(Invitrogen Corporation), following the manufacturer's protocol, toproduce pKR906 (SEQ ID NO:115).

Plasmid pKR906 was digested with NotI, and the fragment containing theEuglena gracilis delta-9 elongase was cloned into plasmid pKR132 (SEQ IDNO:116; which is described in PCT Publication No. WO 2004/071467) toproduce pKR953 (SEQ ID NO:117).

Mortierella Alpina Delta-5 Desaturase (MaD5):

Vector pKR287 (SEQ ID NO:118; which is described in PCT Publication No.WO 2004/071467, published Aug. 26, 2004; the contents of which arehereby incorporated by reference), contains the Mortierella alpinadelta-5 desaturase (MaD5; SEQ ID NO:119, which is described in U.S. Pat.No. 6,075,183 and PCT Publication Nos. WO 2004/071467 and WO2005/047479, the contents of which are hereby incorporated byreference), flanked by the soybean glycinin Gy1 promoter and the pealeguminA2 3′ termination region (Gy1/MaD5/legA2 cassette). Vector pKR287was digested with SbfI/BsiWI, and the fragment containing theGy1/MaD5/legA2 cassette was cloned into the SbfI/BsiWI fragment ofpKR277 (SEQ ID NO:120; which is described in PCT Publication No. WO2004/071467, the contents of which are hereby incorporated by reference)to produce pK952 (SEQ ID NO:121).

Vector pKR457 (SEQ ID NO:122), which was previously described in PCTPublication No. WO 2005/047479 (the contents of which are herebyincorporated by reference), contains a NotI site flanked by the Kunitzsoybean Trypsin Inhibitor (KTi) promoter (Jofuku et al., Plant Cell1:1079-1093 (1989)) and the KTi 3′ termination region, the isolation ofwhich is described in U.S. Pat. No. 6,372,965, followed by the soyalbumin transcription terminator, which was previously described in PCTPublication No. WO 2004/071467 (Kti/NotI/Kti3′Salb3′ cassette). Througha number of sub-cloning steps, sequences containing Asp718 restrictionsites were added to the 5′ and 3′ ends of the Kti/NotI/Kti3′Salb3′cassette to produce SEQ ID NO:123.

Pavlova lutheri Delta-8 Desaturase (PavD8):

Pavlova lutheri (CCMP459) was obtained from the Culture of MarinePhytoplankton (CCMP, West Boothbay Harbor, Me.) and grown in 250 mLflasks containing 50 mL of F/2-Si medium (made using F/2 Family MediumKit-KIT20F2 and Filtered Seqwater-SEA2 from CCMP) at 26° C. with shakingat 150 rpm. Cultures were transferred to new medium on a weekly basisusing a 1:4 (old culture:new medium) dilution.

Cultures from 28 flasks (1400 mL) were combined, and cells were pelletedby centrifugation at 1,800×g for 10 min, washed once with water, andre-centrifuged. Total RNA was extracted from the resulting pellet usingthe RNA STAT-60™ reagent (TEL-TEST, Inc., Friendswood, Tex.), followingthe manufacturer's protocol. In this way, 2.6 mg of total RNA (2.6mg/mL) were obtained from the pellet. The mRNA was isolated from 1.25 mgof total RNA using the mRNA Purification Kit (Amersham Biosciences,Piscataway, N.J.), following the manufacturer's protocol. In this way,112 μg of mRNA were obtained.

cDNA was synthesized from 224 ng of mRNA using the SuperScript™First-Strand Synthesis System for RT-PCR Kit (Invitrogen™ LifeTechnologies, Carlsbad, Calif.) with the provided oligo(dT) primer,according to the manufacturer's protocol. After RNase H treatment as perthe protocol, the Pavlova lutheri delta-8 desaturase (PavD8; SEQ IDNO:124; which is described in U.S. patent application Ser. No.11/737,772 (filed Apr. 20, 2007; Attorney Docket No. BB-1566) thecontents of which are hereby incorporated by reference) was amplifiedfrom the resulting cDNA with oligonucleotide primers PvDES5′Not-1 (SEQID NO:125) and PvDES3′Not-1 (SEQ ID NO:126) using the conditionsdescribed below.

cDNA (2 μL) from the reaction described above was combined with 50 μmolof PvDES5′Not-1 (SEQ ID NO:125), 50 μmol of PvDES3′Not-1 (SEQ IDNO:126), 1 μL of PCR nucleotide mix (10 mM, Promega, Madison, Wis.), 5μL of 10×PCR buffer (Invitrogen Corporation), 1.5 μL of MgCl₂ (50 mM,Invitrogen Corporation), 0.5 μL of Taq polymerase (InvitrogenCorporation), and water to 50 μL. The reaction conditions were 94° C.for 3 min followed by 35 cycles of 94° C. for 45 sec, 55° C. for 45 sec,and 72° C. for 1 min. The PCR was finished at 72° C. for 7 min, and thenheld at 4° C. The PCR reaction was analyzed by agarose gelelectrophoresis on 5 μL, and a DNA band with molecular weight around 1.3kb was observed. The remaining product was separated by agarose gelelectrophoresis, and the DNA was purified using the Zymoclean™ Gel DNARecovery Kit (Zymo Research, Orange, Calif.), following themanufacturer's protocol.

The PavD8, flanked by NotI sites, was cloned into the NotI site of themodified Kti/NotI/Kti3′Salb3′ cassette (SEQ ID NO:123), and then the DNAfragment was digested with Asp718 and cloned into the SbfI site ofpKR952 (SEQ ID NO:121) to produce pKR970 (SEQ ID NO:127).

Plasmid pKR953 (SEQ ID NO:117) was digested with PstI, and the fragmentcontaining the Euglena gracilis delta-9 elongase was cloned into theSbfI site of pKR970 (SEQ ID NO:127) to produce pKR973 (SEQ ID NO:128,FIG. 15B).

In this way, the Pavlova lutheri delta-8 desaturase could beco-expressed with the Mortierella alpina delta-5 desaturase and theEuglena gracilis delta-9 elongase behind strong, seed-specificpromoters.

Example 17 Construction of Soybean Expression Vector pKR1064ForCo-Expression of the Euglena gracilis DHA Synthase 1 (EgDHAsyn1) withthe Saprolegnia diclina Delta-17 Desaturase (SdD17)

The present Example describes construction of a soybean vector forco-expression of EgDHAsyn1 with SdD17 and the acetolactate synthase(ALS) selectable marker.

The PstI fragment, containing the Ann/Sdd17/BD30 cassette from pKR271(SEQ ID NO:129; which is described in PCT Publication No. WO 2004/071467and the contents of which are hereby incorporated by reference), wascloned into the SbfI site of pKR226 (SEQ ID NO:130; which is alsodescribed in PCT Publication No. WO 2004/071467) to produce vectorpKR886r (SEQ ID NO:131). In this way, the Saprolegnia diclina delta-17desaturase (SdD17) was cloned behind the annexin promoter which isstrong and seed specific.

The SbfI fragment of pKR1057 (SEQ ID NO:109), containing theβcon/EgDHAsyn1/Phas3′ cassette, was cloned into the SbfI site of pKR886r(SEQ ID NO:131), containing SdD17, to produce vector pKR1064 (SEQ IDNO:132). A schematic depiction of pKR1064 is shown in FIG. 15C.

Example 18 Construction of Soybean Expression Vector pKR1133ForCo-Expression of the Euglena gracilis DHA Synthase 1 (EgDHAsyn1) Withthe Euglena gracilis Delta-9 Elongase (EgD9elo) and the Mortierellaalpina Delta-5 Desaturase (MaD5)

The glycinin Gy1 promoter was PCR amplified from pZBL119 (SEQ ID NO:133;which is described in PCT Publication No. WO 2004/071467 and thecontents of which are hereby incorporated by reference) using primersoSGly-2 (SEQ ID NO:134) and oSGly-3 (SEQ ID NO:135). The resulting PCRfragment was subcloned into the intermediate cloning vector pCR-ScriptAMP SK(+) (Stratagene), according to the manufacturer's protocol, toproduce plasmid pPSgly32 (SEQ ID NO:136).

The PstI/NotI fragment of plasmid pSGly32 (SEQ ID NO:136), containingthe Gy1 promoter, was cloned into the PstI/NotI fragment from plasmidpKR142 (SEQ ID NO:137; which is described in PCT Publication No. WO2004/071467 and the contents of which are hereby incorporated byreference), containing the leguminA2 3′ transcription terminationregion, an ampicillin resistance gene, and bacterial ori, to producepKR264 (SEQ ID NO:138). Thus, vector pKR264 contains a NotI site flankedby the promoter for the glycinin Gy1 gene and the leguminA2 3′transcription termination region (Gy1/NotI/IegA2 cassette).

EgDHAsyn1 was released from pKR1055 (SEQ ID NO:104; Example 15) bydigestion with NotI and was cloned into the NotI site of plasmid pKR264(SEQ ID NO:138), to produce pKR1128 (SEQ ID NO:139).

The NotI fragment of pKS129 (SEQ ID NO:140; which is described in PCTPublication No. WO 2004/071467 and the contents of which are herebyincorporated by reference), containing the MaD5 was cloned into the NotIsite of pKR457 (SEQ ID NO:122; Example 16), to produce pKR606 (SEQ IDNO:141).

Vector pKR606 (SEQ ID NO:141) was digested with BsiWI and after fillingto blunt the ends, the fragment containing the Gy1/MaD5/legA2 cassettewas cloned into the filled NgoMI site of pKR277 (SEQ ID NO:120) toproduce pKR804 (SEQ ID NO:142).

The BsiWI fragment from pKR1128 (SEQ ID NO:139), containing theGy1/EgDHAsyn1/legA2 cassette, was cloned into the BsiWI site of pKR804(SEQ ID NO:142) to produce pKR1130 (SEQ ID NO:143).

Plasmid pKR953 (SEQ ID NO:117) was digested with BsiWI; ends wereblunted by filling; and pKR953 was then digested with BamHI. The filledBsiWI/BamHI fragment of pKR953, containing the Salb/EgD9Elo/Phas3′cassette, was cloned into the PmeI/BamHI sites of pNEB193 (New EnglandBiolabs, Ipswich, Mass.) to produce pKR1131 (SEQ ID NO:144).

Plasmid pKR1131 (SEQ ID NO:144) was digested with PstI and the fragmentcontaining the Euglena gracilis delta-9 elongase was cloned into theSbfI site of pKR1130 (SEQ ID NO:143) to produce pKR1133 (SEQ ID NO:145,FIG. 15D).

In this way, the Euglena gracilis DHA synthase 1 could be co-expressedwith the Mortierella alpina delta-5 desaturase and the Euglena gracilisdelta-9 elongase behind strong, seed-specific promoters.

Example 19 Construction of Soybean Expression Vector pKR1105ForCo-Expression of the Euglena gracilis DHA Synthase 1 C20 Elongase Domain(EgDHAsyn1C20EloDom1) with the Schizochytrium aggregatum Delta-4Desaturase (SaD4)

The βcon/NotI/Phas cassette was PCR amplified from pKS123 (SEQ IDNO:146; which is described in PCT Publication No. WO 2004/071467 and thecontents of which are hereby incorporated by reference) using primersoKti5 (SEQ ID NO:147) and oKti6 (SEQ ID NO:148). The resulting PCRfragment was digested with BsiWI and cloned into the BsiWI site ofpKR124 (SEQ ID NO:149; which is described in PCT Publication No. WO2004/071467 and the contents of which are hereby incorporated byreference), containing the bacterial origin of replication andselection, to produce plasmid pKR193 (SEQ ID NO:150).

EgDHAsyn1C20Elodom1 was released from pHD16 (SEQ ID NO:51; Example 10)by digestion with NotI and was cloned into the NotI site of plasmidpKR193 (SEQ ID NO:150) to produce pKR1103 (SEQ ID NO:151).

The BsiWI fragment, containing the EgDHAsyn1C20Elodom1, was releasedfrom pKR1103 (SEQ ID NO:151) and was cloned into the BsiWI site ofpKR226 (SEQ ID NO:130; Example 17) to produce vector pKR1104 (SEQ IDNO:152).

Vector pKR300 (SEQ ID NO:153; which is described in PCT Publication No.WO 2004/071467, published Aug. 26, 2004; the contents of which arehereby incorporated by reference), contains the Schizochytriumaggregatum delta-4 desaturase (SaD4), which is described in U.S. Pat.No. 7,045,683 and PCT Publication No WO 02/090493, the contents of whichare hereby incorporated by reference), flanked by the NotI restrictionsites. The AscI site present within the SaD4 was removed withoutaffecting the corresponding amino acid sequence to produce a newsequence (SEQ ID NO:154) which remains flanked by the NotI sites. TheNotI fragment (SEQ ID NO:154) was cloned into the NotI site of plasmidpKR457 (SEQ ID NO:122; Example 16) to produce pKR1102 (SEQ ID NO:155).

Plasmid pKR1102 (SEQ ID NO:155) was digested with PstI, and the fragmentcontaining the SaD4 was cloned into the SbfI site of pKR1104 (SEQ IDNO:152) to produce pKR1105 (SEQ ID NO:156; FIG. 16A). In this way, theEuglena gracilis DHA synthase 1 C20 elongase domain could beco-expressed with the Schizochytrium aggregatum delta-4 desaturasebehind strong, seed-specific promoters.

Example 20 Construction of Soybean Expression Vector pKR1134 ForExpression of the Euglena gracilis DHA Synthase 1 C20 ElongaseDomain/Schizochytrium aggregatum Delta-4 Desaturase Fusion(EgDHAsyn1C20EloDom3-SaD4)

EgDHAsyn1C20EloDom3 was amplified from pKR1091 with oligonucleotideprimers EgEPAEloDom-5 (SEQ ID NO:43) and oEUGsyn6-4 (SEQ ID NO:157)using the Phusion™ High-Fidelity DNA Polymerase (Cat. No. F553S,Finnzymes Oy, Finland) following the manufacturer's protocol. Theresulting DNA fragment was cloned into the pCR-Blunt® cloning vectorusing the Zero Blunt® PCR Cloning Kit (Invitrogen Corporation),following the manufacturer's protocol, to produce pKR1107 (SEQ IDNO:158).

Plasmid pKR1107 (SEQ ID NO:158) was digested with NotI, and the fragmentcontaining the EgDHAsyn1C20EloDom3 was religated to form pKR1112 (SEQ IDNO:159).

The XbaI/PstI DNA fragment from pKR1112 (SEQ ID NO:159), containingEgDHAsyn1C20EloDom3, was cloned into the XbaI/SbfI DNA fragment frompKR1068 (SEQ ID NO:75; Example 11), containing the SaD4, to producepKR1115 (SEQ ID NO:160). In this way, the EgDHAsyn1C20Elodom3-SaD4 wasre-created without an internal SbfI site but codes for an identicalamino acid sequence as that described in Example 11.

EgDHAsyn1C20Elodom3-SaD4 was released from pKR1115 (SEQ ID NO:160) bydigestion with NotI and was cloned into the NotI site of plasmid pKR1104(SEQ ID NO:152), containing an ALS selectable marker, to produce pKR1134(SEQ ID NO:161; FIG. 16B).

Example 21 Construction of Soybean Expression Vector pKR1095ForCo-Expression of the Tetruetreptia pomguetensis CCMP1491 Delta-8Desaturase (TpomD8) with the Saprolegnia diclina Delta-17 Desaturase(SdD17)

The present Example describes construction of a soybean vector forco-expression of TpomD8 with SdD17 and a hygromycin phosphotransferaseselectable marker (hpt).

Tetruetreptia pomquetensis CCMP1491 cells (from 1 liter of culture) werepurchased from the Provasoli-Guillard National Center for Culture ofMarine Phytoplakton (CCMP) (Bigelow Laboratory for Ocean Sciences, WestBoothbay Harbor, Me.). Total RNA was isolated using the trizol reagent(Invitrogen, Carlsbad, Calif.), according to the manufacturer'sprotocol. The cell pellet was resuspended in 0.75 mL of trizol reagent,mixed with 0.5 mL of 0.5 mm glass beads, and homogenized in a Biospecmini beadbeater (Bartlesville, Okla.) at the highest setting for 3 min.The mixture was centrifuged in an Eppendorf centrifuge for 30 sec at14,000 rpm to remove debri and glass beads. Supernatant was extractedwith 150 μL of 24:1 chloroform:isoamy alcohol. The upper aqueous phasewas used for RNA isolation.

For RNA isolation, the aqueous phase was mixed with 0.375 mL ofisopropyl alcohol and allowed to incubate at room temperature for 5 min.Precipitated RNA was collected by centrifugation at 8,000 rpm and keptat 4° C. for 5 min. The pellet was washed once with 0.7 mL of 80%ethanol and air dried. Thus, 95 μg of total RNA were obtained fromTetruetreptia pomquetensis CCMP1491.

Total RNA (0.95 μg of total RNA in 1 μL) was used as template tosynthesize double stranded cDNA. The Creator™ SMART™ cDNA LibraryConstruction Kit from BD Bioscience Clontech (Palo Alto, Calif.) wasused. Total RNA (1 μL) was mixed with 1 μL of SMART IV oligonucleotide(SEQ ID NO:181) 1 μL of the Adaptor Primer from Invitrogen 3′-RACE kit(SEQ ID NO:182), and 2 μL of water. The mixture was heated to 75° C. for5 min and then cooled on ice for 5 min. To the mixture was added: 2 μLof 5× first strand buffer, 1 μL 20 mM DTT, 1 μL of dNTP mix (10 mM eachof dATP, dCTP, dGTP and dTTP), and 1 μL of PowerScript reversetranscriptase. The sample was incubated at 42° C. for 1 h. The resultingfirst strand cDNAs were then used as templates for amplification.

The Tetruetreptia pomquetensis CCMP1491 delta-8 desaturase (TpomD8; SEQID NO:162; which is described in U.S. patent application Ser. No.11/876,115 (filed Oct. 22, 2007; Attorney Docket No. BB-1574) thecontents of which are hereby incorporated by reference) was amplifiedfrom the cDNA with oligonucleotide primers TpomNot-5 (SEQ ID NO:163) andTpomNot-3 (SEQ ID NO:164) using Taq polymerase (Invitrogen Corporation)following the manufacturer's protocol.

Tetruetreptia pomquetensis CCMP1491 cDNA (1 μL) was combined with 50μmol of TpomNot-5 (SEQ ID NO:163), 50 μmol of TpomNot-3 (SEQ ID NO:164),1 μL of PCR nucleotide mix (10 mM, Promega, Madison, Wis.), 5 μL of10×PCR buffer (Invitrogen Corporation), 1.5 μL of MgCl₂ (50 mM,Invitrogen Corporation), 0.5 μL of Taq polymerase (InvitrogenCorporation) and water to 50 μL. The reaction conditions were 94° C. for3 min followed by 35 cycles of 94° C. for 45 sec, 55° C. for 45 sec and72° C. for 1 min. The PCR was finished at 72° C. for 7 min and then heldat 4° C. 5 μL of the PCR reaction were analyzed by agarose gelelectrophoresis, and a DNA band with molecular weight around 1.3 kb wasobserved.

The remaining product was separated by agarose gel electrophoresis, andthe DNA was purified using the Zymoclean™ Gel DNA Recovery Kit (ZymoResearch, Orange, Calif.), following the manufacturer's protocol. Theresulting DNA was cloned into the pGEM®-T Easy Vector (Promega),following the manufacturer's protocol, to produce pLF114-10 (SEQ IDNO:165).

TpomD8 was released from pLF114-10 (SEQ ID NO:165) by digestion withNotI and was cloned into the NotI site of plasmid pKR179 (SEQ ID NO:108;Example 15) to produce pKR1002 (SEQ ID NO:166).

The PstI fragment of pKR1002 (SEQ ID NO:166), containing theβcon/TpomD8/Phas3′ cassette was cloned into the SbfI site of pKR328 (SEQID NO:110; Example 15), containing the SdD17, to produce vector pKR1095(SEQ ID NO:167). A schematic depiction of pKR1095 is shown in FIG. 16C.

Example 22 Construction of Soybean Expression Vector pKR1132ForCo-Expression of the Tetruetreptia pomquetensis CCMP1491 Delta-8Desaturase (TpomD8) with the Euglena gracilis Delta-9 Elongase (EgD9elo)and the Mortierella alpina Delta-5 Desaturase (MaD5)

TPomD8 was released from pLF114-10 (SEQ ID NO:165; Example 21) bydigestion with NotI and was cloned into the NotI site of plasmid pKR264(SEQ ID NO:138; Example 18) to produce pKR1127 (SEQ ID NO:168).

The BsiWI fragment from pKR1127 (SEQ ID NO:168), containing theGy1/TPomD8/legA2 cassette, was cloned into the BsiWI site of pKR804 (SEQID NO:142; Example 18) to produce pKR1129 (SEQ ID NO:169).

Plasmid pKR1131 (SEQ ID NO:144; Example 18) was digested with PstI, andthe fragment containing the Euglena gracilis delta-9 elongase was clonedinto the SbfI site of pKR1129 (SEQ ID NO:169) to produce pKR1132 (SEQ IDNO:170, FIG. 16D). In this way, the Tetruetreptia pomquetensis delta-8desaturase could be co-expressed with the Mortierella alpina delta-5desaturase and the Euglena gracilis delta-9 elongase behind strong,seed-specific promoters.

Example 23 Construction of Soybean Expression Vector KS373 forExpression of a Euglena gracilis Delta-9 Elongase/Euglena gracilis DHASynthase 1 Linker/Pavlova lutheri Delta-8 Desaturase Fusion(EgD9elo-EgDHAsyn1Link-PavD8)

An in-frame fusion between the Euglena gracilis delta-9 elongase(EgD9elo; Example 16; SEQ ID NO:112), the Euglena gracilis DHA synthase1 proline-rich linker (EgDHAsyn1Link; SEQ ID NO:171; described inExample 6 and shown in FIG. 6), and the Pavlova lutheri delta-8desaturase (PavD8; Example 16; SEQ ID NO:124) was constructed using theconditions described below.

An initial in-frame fusion between the EgD9elo and the EgDHAsyn1Link(EgD9elo-EgDHAsyn1Link) was made, flanked by a NcoI site at the 5′ endand a NotI site at the 3′ end, by PCR amplification. EgD9elo (SEQ IDNO:112) was amplified with oligonucleotides MWG507 (SEQ ID NO:172) andMWG509 (SEQ ID NO:173), using the Phusion™ High-Fidelity DNA Polymerase(Cat. No. F553S, Finnzymes Oy, Finland), following the manufacturer'sprotocol. EgDHAsyn1Link (SEQ ID NO:171) was amplified in a similar waywith oligonucleotides MWG510 (SEQ ID NO:174) and MWG511 (SEQ ID NO:175).The two resulting PCR products were combined and re-amplified usingMWG507 (SEQ ID NO:172) and MWG511 (SEQ ID NO:175) to formEgD9elo-EgDHAsyn1Link. The sequence of the EgD9elo-EgDHAsyn1Link isshown in SEQ ID NO:176. EgD9elo-EgDHAsyn1Link does not contain anin-frame stop codon upstream of the NotI site at the 3′ end, andtherefore, a DNA fragment cloned into the NotI site can give rise to anin-frame fusion with the EgD9elo-EgDHAsyn1Link.

Plasmid KS366 (SEQ ID NO:177) contains unique NcoI and NotI restrictionsites, flanked by the promoter for the α′ subunit of β-conglycinin(Beachy et al., EMBO J. 4:3047-3053 (1985)) and the 3′ transcriptiontermination region of the phaseolin gene (Doyle et al., J. Biol. Chem.261:9228-9238 (1986)). Other than the replacement of the unique NotIsite in pKR72 (SEQ ID NO:105) with a unique NcoI/NotI multiple cloningsite, the Bcon/NcoINotI/Phas3′ cassette in KS366 is identical to thatfound in pKR72 (SEQ ID NO:105), except that the flanking HindIII siteswere replaced by BamHI sites. The Bcon/NcoINotI/Phas3′ cassette of KS366was cloned into the BamHI site of pBluescript II SK(+) vector(Stratagene).

The NcoI/NotI DNA fragment, containing EgD9elo-EgDHAsyn1Link (SEQ IDNO:176), was cloned into the NcoI/NotI DNA fragment from KS366 (SEQ IDNO:177), containing the promoter for the α′ subunit of β-conglycinin, toproduce KS366-EgD9elo-EgDHAsyn1Link (SEQ ID NO:178).

The NotI fragment containing PavD8 (generated as described in Example16) was cloned into the NotI fragment of KS366-EgD9elo-EgDHAsyn1Link(SEQ ID NO:178) to produce KS373 (SEQ ID NO:179; FIG. 17).

Example 24 Construction of Alternate Soybean Expression Vectors forExpression of DHA Synthases, C20 Elongase Domains, Delta-4 DesaturaseDomains, Synthetic C20 Elongase/Delta-4 Desaturase Fusion Proteins andOther Synthetic Elongase/Desaturase Fusion Proteins

In addition to the genes, promoters, terminators and gene cassettesdescribed herein, one skilled in the art can appreciate that otherpromoter/gene/terminator cassette combinations can be synthesized in away similar to, but not limited to, that described herein for expressionof EgDHAsyn1. Similarly, it may be desirable to express other PUFA genes(such as those described in Table 23), for co-expression with any of theDHA synthases of the present invention or DHA synthase domains (i.e.,C20 elongase domain or delta-4 desaturase domain expressedindividually). Additionally, synthetic fusions between an elongasedomain and a desaturase domain separated by a suitable linker regioncould be made and expressed. For instance, a synthetic fusion between aC20 elongase (or C20 elongase domain from a DHA synthase) and a suitabledelta-4 desaturase (or delta-4 desaturase domain from a DHA synthase)could be made and expressed. Alternatively, other elongases ordesaturases could be used such as, but not limited to, the syntheticfusion described herein between a delta-9 elongase and delta-8desaturase separated by a linker from a DHA synthase (i.e., Example 23).

For instance, PCT Publication Nos. WO 2004/071467 and WO 2004/071178describe the isolation of a number of promoter and transcriptionterminator sequences for use in embryo-specific expression in soybean.Furthermore, PCT Publication Nos. WO 2004/071467, WO 2005/047479 and WO2006/012325 describe the synthesis of multiple promoter/gene/terminatorcassette combinations by ligating individual promoters, genes, andtranscription terminators together in unique combinations. Generally, aNotI site flanked by the suitable promoter (such as those listed in, butnot limited to, Table 21) and a transcription terminator (such as thoselisted in, but not limited to, Table 22) is used to clone the desiredgene. NotI sites can be added to a gene of interest such as those listedin, but not limited to, Table 23 using PCR amplification witholigonucleotides designed to introduce NotI sites at the 5′ and 3′ endsof the gene. The resulting PCR product is then digested with NotI andcloned into a suitable promoter/NotI/terminator cassette.

In addition, PCT Publication Nos. WO 2004/071467, WO 2005/047479 and WO2006/012325 describe the further linking together of individual genecassettes in unique combinations, along with suitable selectable markercassettes, in order to obtain the desired phenotypic expression.Although this is done mainly using different restriction enzymes sites,one skilled in the art can appreciate that a number of techniques can beutilized to achieve the desired promoter/gene/transcription terminatorcombination. In so doing, any combination of embryo-specificpromoter/gene/transcription terminator cassettes can be achieved. Oneskilled in the art can also appreciate that these cassettes can belocated on individual DNA fragments or on multiple fragments whereco-expression of genes is the outcome of co-transformation of multipleDNA fragments.

TABLE 21 Seed-specific Promoters Promoter Organism Promoter Referenceβ-conglycinin α′-subunit soybean Beachy et al., EMBO J. 4: 3047-3053(1985) kunitz trypsin inhibitor soybean Jofuku et al., Plant Cell 1:1079-1093 (1989) Annexin soybean WO 2004/071467 glycinin Gy1 soybean WO2004/071467 albumin 2S soybean U.S. Pat. No. 6,177,613 legumin A1 peaRerie et al., Mol. Gen. Genet. 225: 148-157 (1991) β-conglycininβ-subunit soybean WO 2004/071467 BD30 (also called P34) soybean WO2004/071467 legumin A2 pea Rerie et al., Mol. Gen. Genet. 225: 148-157(1991)

TABLE 22 Transcription Terminators Transcription Terminator OrganismReference phaseolin 3′ bean WO 2004/071467 kunitz trypsin inhibitor 3′soybean WO 2004/071467 BD30 (also called P34) 3′ soybean WO 2004/071467legumin A2 3′ pea WO 2004/071467 albumin 2S 3′ soybean WO 2004/071467

TABLE 23 PUFA Biosynthetic Pathway Genes Gene Organism Reference delta-6desaturase Saprolegnia diclina WO 2002/081668 delta-6 desaturaseMortierella alpina U.S. Pat. No. 5,968,809 elongase Mortierella alpinaWO 2000/12720 U.S. Pat. No. 6,403,349 delta-5 desaturase Mortierellaalpina U.S. Pat. No. 6,075,183 delta-5 desaturase Saprolegnia diclina WO2002/081668 delta-5 desaturase Peridinium sp. U.S. Patent ApplicationNo. 11/748637 delta-5 desaturase Euglena gracilis U.S. PatentApplication No. 11/748629 delta-15 desaturase Fusarium moniliforme WO2005/047479 delta-17 desaturase Saprolegnia diclina WO 2002/081668elongase Thraustochytrium WO 2002/08401 aureum U.S. Pat. No. 6,677,145elongase Pavlova sp. Pereira et al., Biochem. J. 384: 357-366 (2004)delta-4 desaturase Schizochytrium WO 2002/090493 aggregatum U.S. Pat.No. 7,045,683 delta-4 desaturase Isochrysis galbana WO 2002/090493 U.S.Pat. No. 7,045,683 delta-4 desaturase Thraustochytrium WO 2002/090493aureum U.S. Pat. No. 7,045,683 delta-4 desaturase Euglena gracilis U.S.Patent Application No. 10/552,127 delta-9 elongase Isochrysis galbana WO2002/077213 delta-9 elongase Euglena gracilis U.S. Patent ApplicationNo. 11/601,563 delta-9 elongase Eutreptiella sp. U.S. Patent ApplicationCCMP389 No. 11/601,564 delta-9 elongase Euglena anabaena Pending delta-8desaturase Euglena gracilis WO 2000/34439 U.S. Pat. No. 6,825,017 WO2004/057001 WO 2006/012325 delta-8 desaturase Acanthamoeba Sayanova etal., FEBS castellanii Lett. 580: 1946-1952 (2006) delta-8 desaturasePavlova salina WO 2005/103253 delta-8 desaturase Pavlova lutheri U.S.Patent Application No. 11/737772 delta-8 desaturase Tetruetreptia U.S.Patent Application pomquetensis No. 11/876115 CCMP1491 delta-8desaturase Eutreptiella sp. U.S. Patent Application CCMP389 No.11/876115 delta-8 desaturase Eutreptiella U.S. Patent Applicationcf_gymnastica No. 11/876115 CCMP1594 delta-8 desaturase Euglena anabaenaPending

Example 25 Production and Model System Transformation of Somatic SoybeanEmbryo Cultures with Soybean Expression Vectors Culture Conditions:

Soybean embryogenic suspension cultures (cv. Jack) are maintained in 35mL liquid medium SB196 (infra) on a rotary shaker, 150 rpm, 26° C. withcool white fluorescent lights on 16:8 hr day/night photoperiod at lightintensity of 60-85 μE/m2/s. Cultures are subcultured every 7 days to twoweeks by inoculating approximately 35 mg of tissue into 35 mL of freshliquid SB196 (the preferred subculture interval is every 7 days).

Soybean embryogenic suspension cultures are transformed with the soybeanexpression plasmids by the method of particle gun bombardment (Klein etal., Nature 327:70 (1987)) using a DuPont Biolistic PDS1000/HEinstrument (helium retrofit) for all transformations.

Soybean Embryogenic Suspension Culture Initiation:

Soybean cultures are initiated twice each month with 5-7 days betweeneach initiation. Pods with immature seeds from available soybean plantsare picked 45-55 days after planting. Seeds are removed from the podsand placed into a sterilized magenta box. The soybean seeds aresterilized by shaking them for 15 min in a 5% Clorox solution with 1drop of Ivory soap (i.e., 95 mL of autoclaved distilled water plus 5 mLClorox and 1 drop of soap, mixed well). Seeds are rinsed using 2 1-literbottles of sterile distilled water and those less than 4 mm are placedon individual microscope slides. The small end of the seed is cut andthe cotyledons are pressed out of the seed coat. When cultures are beingprepared for production transformation, cotyledons are transferred toplates containing SB1 medium (25-30 cotyledons per plate). Plates arewrapped with fiber tape and are maintained at 26° C. with cool whitefluorescent lights on 16:8 h day/night photoperiod at light intensity of60-80 μE/m2/s for eight weeks, with a media change after 4 weeks. Whencultures are being prepared for model system experiments, cotyledons aretransferred to plates containing SB199 medium (25-30 cotyledons perplate) for 2 weeks, and then transferred to SB1 for 2-4 weeks. Light andtemperature conditions are the same as described above. After incubationon SB1 medium, secondary embryos are cut and placed into SB196 liquidmedia for 7 days.

Preparation of DNA for Bombardment:

Either an intact plasmid or a DNA plasmid fragment containing the genesof interest and the selectable marker gene are used for bombardment.Fragments from soybean expression plasmids, the construction of which isdescribed herein, are obtained by gel isolation of digested plasmids. Ineach case, 100 μg of plasmid DNA is used in 0.5 mL of the specificenzyme mix described below. Plasmids are digested with AscI (100 units)in NEBuffer 4 (20 mM Tris-acetate, 10 mM magnesium acetate, 50 mMpotassium acetate, 1 mM dithiothreitol, pH 7.9), 100 μg/mL BSA, and 5 mMbeta-mercaptoethanol at 37° C. for 1.5 hr. The resulting DNA fragmentsare separated by gel electrophoresis on 1% SeaPlaque GTG agarose(BioWhitaker Molecular Applications), and the DNA fragments containinggene cassettes are cut from the agarose gel. DNA is purified from theagarose using the GELase digesting enzyme following the manufacturer'sprotocol.

A 50 μL aliquot of sterile distilled water containing 3 mg of goldparticles (3 mg gold) is added to 30 μL of a 10 ng/μL DNA solution(either intact plasmid or DNA fragment prepared as described herein), 25μL 5M CaCl₂, and 20 μL of 0.1 M spermidine. The mixture is shaken 3 minon level 3 of a vortex shaker and spun for 10 sec in a bench microfuge.The supernatant is removed, followed by a wash with 400 μL 100% ethanoland another brief centrifugation. The 400 ul ethanol is removed, and thepellet is resuspended in 40 μL of 100% ethanol. Five μL of DNAsuspension is dispensed to each flying disk of the Biolistic PDS1000/HEinstrument disk. Each 5 μL aliquot contains approximately 0.375 mg goldper bombardment (e.g., per disk).

For model system transformations, the protocol is identical except for afew minor changes (i.e., 1 mg of gold particles is added to 5 μL of a 1μg/μL DNA solution; 50 μL of a 2.5M CaCl₂ is used; and the pellet isultimately resuspended in 85 μL of 100% ethanol thus providing 0.058 mgof gold particles per bombardment).

Tissue Preparation and Bombardment with DNA:

Approximately 150-200 mg of seven day old embryogenic suspensioncultures is placed in an empty, sterile 60×15 mm petri dish, and thedish is covered with plastic mesh. The chamber is evacuated to a vacuumof 27-28 inches of mercury, and tissue is bombarded one or two shots perplate with membrane rupture pressure set at 1100 PSI. Tissue is placedapproximately 3.5 inches from the retaining/stopping screen. Modelsystem transformation conditions are identical except 100-150 mg ofembryogenic tissue is used; rupture pressure is set at 650 PSI; andtissue is placed approximately 2.5 inches from the retaining screen.

Selection of Transformed Embryos:

Transformed embryos are selected either using hygromycin (when thehygromycin B phosphotransferase (HPT) gene is used as the selectablemarker) or chlorsulfuron (when the acetolactate synthase (ALS) gene isused as the selectable marker).

Following bombardment, the tissue is placed into fresh SB196 media andcultured as described above. Six to eight days post-bombardment, theSB196 is exchanged with fresh SB196 containing either 30 mg/L hygromycinor 100 ng/mL chlorsulfuron, depending on the selectable marker used. Theselection media is refreshed weekly. Four to six weeks post-selection,green, transformed tissue is observed growing from untransformed,necrotic embryogenic clusters.

Embryo Maturation:

For production transformations, isolated, green tissue is removed andinoculated into multiwell plates to generate new, clonally propagated,transformed embryogenic suspension cultures. Transformed embryogenicclusters are cultured for four-six weeks in multiwell plates at 26° C.in SB196 under cool white fluorescent (Phillips cool white EconowattF40/CW/RS/EW) and Agro (Phillips F40 Agro) bulbs (40 watt) on a 16:8 hrphotoperiod with light intensity of 90-120 μE/m²s. After this time,embryo clusters are removed to a solid agar media, SB166, for one-twoweeks and then subcultured to SB103 medium for 3-4 weeks to matureembryos. After maturation on plates in SB103, individual embryos areremoved from the clusters, dried, and screened for alterations in theirfatty acid compositions as described supra.

For model system transformations, embryos are matured in soybeanhistodifferentiation and maturation liquid medium (SHaM liquid media;Schmidt et al., Cell Biology and Morphogenesis 24:393 (2005)), using amodified procedure. Briefly, after 4 weeks of selection in SB196, asdescribed above, embryo clusters are removed to 35 mL of SB228 (SHaMliquid media) in a 250 mL Erlenmeyer flask. Tissue is maintained in SHaMliquid media on a rotary shaker at 130 rpm and 26° C., with cool whitefluorescent lights on a 16:8 hr day/night photoperiod at a lightintensity of 60-85 μE/m2/s for 2 weeks as embryos matured. Embryos grownfor 2 weeks in SHaM liquid media are equivalent in size and fatty acidcontent to embryos cultured on SB166/SB103 for 5-8 weeks.

After maturation in SHaM liquid media, individual embryos are removedfrom the clusters, dried, and screened for alterations in their fattyacid compositions as described supra.

Media Recipes:

SB 196 - FN Lite Liquid Proliferation Medium (per liter) MS FeEDTA -100x Stock 1 10 mL MS Sulfate - 100x Stock 2 10 mL FN Lite Halides -100x Stock 3 10 mL FN Lite P, B, Mo - 100x Stock 4 10 mL B5 vitamins (1mL/L) 1.0 mL 2,4-D (10 mg/L final concentration) 1.0 mL KNO₃ 2.83 gm(NH₄)₂SO₄ 0.463 gm asparagine 1.0 gm sucrose (1%) 10 gm pH 5.8

FN Lite Stock Solutions

Stock Number 1000 mL 500 mL 1 MS Fe EDTA 100x Stock Na₂EDTA* 3.724 g1.862 g FeSO₄—7H₂O 2.784 g 1.392 g 2 MS Sulfate 100x stock MgSO₄—7H₂O37.0 g 18.5 g MnSO₄—H₂O 1.69 g 0.845 g ZnSO₄—7H₂O 0.86 g 0.43 gCuSO₄—5H₂O 0.0025 g 0.00125 g 3 FN Lite Halides 100x Stock CaCl₂—2H₂O30.0 g 15.0 g KI 0.083 g 0.0715 g CoCl₂—6H₂O 0.0025 g 0.00125 g 4 FNLite P, B, Mo 100x Stock KH₂PO₄ 18.5 g 9.25 g H₃BO₃ 0.62 g 0.31 gNa₂MoO₄—2H₂O 0.025 g 0.0125 g *Add first, dissolve in dark bottle whilestirring

SB1 Solid Medium (Per Liter)

-   -   1 package MS salts (Gibco/BRL—Cat. No. 11117-066)    -   1 mL B5 vitamins 1000× stock    -   31.5 g glucose    -   2 mL 2,4-D (20 mg/L final concentration)    -   pH 5.7    -   8g TC agar

SB199 Solid Medium (Per Liter)

-   -   1 package MS salts (Gibco/BRL—Cat. No. 11117-066)    -   1 mL B5 vitamins 1000× stock    -   30 g Sucrose    -   4 ml 2,4-D (40 mg/L final concentration)    -   pH 7.0    -   2 gm Gelrite

SB 166 Solid Medium (Per Liter)

-   -   1 package MS salts (Gibco/BRL—Cat. No. 11117-066)    -   1 mL B5 vitamins 1000× stock    -   60 g maltose    -   750 mg MgCl₂ hexahydrate    -   5g activated charcoal    -   pH 5.7    -   2g gelrite

SB 103 Solid Medium (Per Liter)

-   -   1 package MS salts (Gibco/BRL—Cat. No. 11117-066)    -   1 mL B5 vitamins 1000× stock    -   60 g maltose    -   750 mg MgCl₂ hexahydrate    -   pH 5.7    -   2g gelrite

SB 71-4 Solid Medium (Per Liter)

-   -   1 bottle Gamborg's B5 salts w/sucrose (Gibco/BRL—Cat. No.        21153-036)    -   pH 5.7    -   5 g TC agar

2,4-D Stock

Obtain premade from Phytotech Cat. No. D 295—concentration 1 mg/mL

B5 Vitamins Stock (Per 100 mL)

Store aliquots at −20° C.

-   -   10 g myo-inositol    -   100 mg nicotinic acid    -   100 mg pyridoxine HCl    -   1 g thiamine        If the solution does not dissolve quickly enough, apply a low        level of heat via the hot stir plate.

SB 228-Soybean Histodifferentiation & Maturation (SHaM) (Per Liter)

DDI H₂O 600 mL FN-Lite Macro Salts for SHaM 10X 100 mL MS Micro Salts1000x 1 mL MS FeEDTA 100x 10 mL CaCl 100x 6.82 mL B5 Vitamins 1000x 1 mLL-Methionine 0.149 g Sucrose 30 g Sorbitol 30 g Adjust volume to 900 mLpH 5.8 Autoclave Add to cooled media (≦30° C.): *Glutamine (finalconcentration 30 mM) 4% 110 mL *Note: Final volume will be 1010 mL afterglutamine addition. Since glutamine degrades relatively rapidly, it maybe preferable to add immediately prior to using media. Expiration 2weeks after glutamine is added; base media can be kept longer withoutglutamine.

FN-Lite Macro for SHAM 10×—Stock #1 (Per Liter)

(NH₄)₂SO₄(ammonium sulfate) 4.63 g KNO₃(potassium nitrate) 28.3 gMgSO₄*7H₂0 (magnesium sulfate heptahydrate)  3.7 g KH₂PO₄(potassiumphosphate, monobasic) 1.85 g Bring to volume Autoclave

MS Micro 1000×—Stock #2 (per 1 liter)

H₃BO₃(boric acid) 6.2 g MnSO₄*H₂O (manganese sulfate monohydrate) 16.9 gZnSO4*7H₂0 (zinc sulfate heptahydrate) 8.6 g Na₂MoO₄*2H₂0 (sodiummolybdate dihydrate) 0.25 g CuSO₄*5H₂0 (copper sulfate pentahydrate)0.025 g CoCl₂*6H₂0 (cobalt chloride hexahydrate) 0.025 g KI (potassiumiodide) 0.8300 g Bring to volume Autoclave

FeEDTA 100×—Stock #3 (Per Liter)

Na₂EDTA* (sodium EDTA) 3.73 g FeSO₄*7H₂0 (iron sulfate heptahydrate)2.78 g Bring to Volume Solution is photosensitive. Bottle(s) should bewrapped in foil to omit light. Autoclave *EDTA must be completelydissolved before adding iron.

Ca 100×—Stock #4 (Per Liter)

CaCl₂*2H₂0 (calcium chloride dihydrate) 44 g Bring to Volume Autoclave

B5 Vitamin 1000×—Stock #5 (Per Liter)

Thiamine*HCl 10 g Nicotinic Acid 1 g Pyridoxine*HCl 1 g Myo-Inositol 100g Bring to Volume Store frozen

4% Glutamine—Stock #6 (Per Liter)

DDI water heated to 30° C. 900 mL L-Glutamine 40 g Gradually add whilestirring and applying low heat. Do not exceed 35° C. Bring to VolumeFilter Sterilize Store frozen* *Note: Warm thawed stock in 31° C. bathto fully dissolve crystals.

Example 26 Chlorsulfuron Selection (ALS) and Plant RegenerationChlorsulfuron (ALS) Selection:

Following bombardment, the tissue is divided between 2 flasks with freshSB196 media and cultured as described in Example 25. Six to seven dayspost-bombardment, the SB196 is exchanged with fresh SB196 containingselection agent of 100 ng/mL chlorsulfuron (chlorsulfuron stock is 1mg/mL in 0.01 N ammonium hydroxide). The selection media is refreshedweekly. Four to six weeks post selection, green, transformed tissue maybe observed growing from untransformed, necrotic embryogenic clusters.Isolated, green tissue is removed and inoculated into multiwell platescontaining SB196, and embryos are matured as described in Example 25.

Regeneration of Soybean Somatic Embryos into Plants:

In order to obtain whole plants from embryogenic suspension cultures,the tissue must be regenerated. Embryos are matured as described inExample 25. After subculturing on medium SB103 for 3 weeks, individualembryos can be removed from the clusters and screened for alterations intheir fatty acid compositions as described herein. It should be notedthat any detectable phenotype, resulting from the expression of thegenes of interest, could be screened at this stage. This would include,but not be limited to, alterations in fatty acid profile, proteinprofile and content, carbohydrate content, growth rate, viability, orthe ability to develop normally into a soybean plant.

Matured individual embryos are desiccated by placing them into an empty,small petri dish (35×10 mm) for approximately 4 to 7 days. The platesare sealed with fiber tape (creating a small humidity chamber).Desiccated embryos are planted into SB71-4 medium where they are left togerminate under the same culture conditions described above. Germinatedplantlets are removed from germination medium and rinsed thoroughly withwater and then are planted in Redi-Earth in 24-cell pack tray, coveredwith clear plastic dome. After 2 weeks the dome is removed, and plantsare hardened off for a further week. If plantlets look hardy, they aretransplanted to 10″ pot of Redi-Earth with up to 3 plantlets per pot.After 10 to 16 weeks, mature seeds are harvested, chipped, and analyzedfor fatty acids.

Example 27 Functional Analysis of the Euglena gracilis and Euglenaanabaena DHA Synthases in Yarrowia lipolytica

Each of the expression vectors described below in Table 24 wastransformed into Yarrowia lipolytica strain Y2224 (a uracil ura3auxotrophic strain of Yarrowia lipolytica) as described in the GeneralMethods above.

Single colonies of transformant Yarrowia lipolytica containing anappropriate Yarrowia expression vector (see Table 24) were grown in 3 mLMM lacking uracil supplemented with 0.2% tergitol at 30° C. for 1 day.After this, 0.05 mL was transferred to 3 mL of the same mediumsupplemented with either no fatty acid or EPA to 0.175 mM. These wereincubated for 16 hr at 30° C., 250 rpm and then pellets were obtained bycentrifugation. Cells were washed once with water, pelleted bycentrifugation and air dried. Pellets were transesterified (Roughan, G.and Nishida, I., Arch. Biochem. Biophys. 276(1):38-46 (1990)) with 500μL of 1% sodium methoxide for 30 min at 50° C. after which 500 μL of 1 Msodium chloride and 100 μL of heptane were added. After thorough mixingand centrifugation, fatty acid methyl esters (FAMEs) were analyzed by GCas described supra (see General Methods).

TABLE 24 Summary of Vectors Transformed Into Yarrowia lipolytica VectorGene SEQ Back- Expressed Ex. FIG. ID Vector bone (SEQ ID NO:) No. No.NO: Comments pBY- pBY1 EgC20elo1 9  7D 39 EgC20elo1 expressed EgC20elo1(SEQ ID as fusion protein NO: 5) pY132 pBY1 EgDHAsyn1 9  8A 40 EgDHAsyn1expressed (SEQ ID as fusion protein NO: 11) pY161 pY159 EgDHAsyn1 9  8B41 EgDHAsyn1 expressed (SEQ ID from gene's NO: 11) translational startsite pY164 pY159 EgDHAsyn2 9  8C 42 EgDHAsyn2 expressed (SEQ ID fromgene's NO: 21) translational start site pY141 pY115 EgDHAsyn1* 10  8D 49Internal Ncol site of (SEQ ID EgDHAsyn1 was NO: 205) removed to yieldEgDHAsyn1*; no further nucleotide changes made pY165 pY159 EaDHAsyn1 1414A 99 EaDHAsyn1 expressed (SEQ ID from gene's NO: 91) translationalstart site pY166 pY159 EaDHAsyn2 14 14B 100 EaDHAsyn2 expressed (SEQ IDfrom gene's NO: 92) translational start site pY167 pY159 EaDHAsyn3 1414C 101 EaDHAsyn3 expressed (SEQ ID from gene's NO: 93) translationalstart site pY168 pY159 EaDHAsyn4 14 14D 102 EaDHAsyn4 expressed (SEQ IDfrom gene's NO: 94) translational start site

The fatty acid profile for Yarrowia expressing pBY-EgC20elo1 showed noelongation of EPA to DPA. The fatty acid profiles, calculated %elongation and calculated % desaturation for the remaining clones areshown in FIG. 18. Percent C20 elongation (% C20 Elong) was calculated bydividing the sum of the weight percent (wt. %) for DPA and DHA by thesum of the wt. % for EPA, DPA and DHA and multiplying by 100 to expressas a %. Similarly, percent delta-4 desaturation (% D4 Desat) wascalculated by dividing the wt. % for DHA by the sum of the wt. % for DPAand DHA and multiplying by 100 to express as a %. Averages are indicatedby Ave. followed by appropriate header.

In summary of FIG. 18, all of the DHA synthases except for EaDHAsyn4functioned as both C20 elongases (elongating EPA to DPA) and as delta-4desaturases (desaturating DPA to DHA) in Yarrowia. EaDHAsyn4, whichcontained a substantially different amino acid sequence at theC-terminus due to a frameshift in the nucleotide sequence, hadconsiderably lower elongation function and no desaturase activity wasdetected. Expressing EgDHAsyn1 in pY132 consistently resulted in higheractivity in Yarrowia when compared to the other EgDHAsyn1 constructs,likely due to the fact that EgDHAsyn1 was expressed as an in-framefusion between some vector sequence, the 5′ UTR of EgDHAsyn1 and theEgDHAsyn1 coding sequence. The resulting fusion created may lead toenhanced activity because of enhanced expression in Yarrowia or becauseof an inherent increase in activity to the enzyme itself. When only thecoding sequence of EgDHAsyn1* is expressed (i.e., with no 5′UTR; seepY141), the activity is higher than when the 5′UTR is present but nottranslated as a fusion (i.e., see pY161). This observation is likely dueto a decrease in expression of EgDHAsyn1 due to the presence of the5′UTR.

Example 28 Functional Analysis of EgDHAsyn1 Independent C20 Elongase andDelta-4 Desaturase Domains and Comparison with Heterologous Fusions

Each of the expression vectors described below in Table 25 (and a vectoronly control) was transformed into Yarrowia lipolytica strain Y2224, asdescribed in Example 27. EPA and/or DPA was fed to the transformedYarrowia cells. A schematic showing the relative domain structure foreach construct in Table 25 is shown in FIG. 21.

TABLE 25 Summary of Vectors Expressed in Yarrowia lipolytica GeneExpressed Ex. FIG. SEQ Vector (SEQ ID NO:) No. No. ID NO: Comments pY141EgDHAsyn1* 10  8D 49 internal NcoI site of EgDHAsyn1 (SEQ ID was removedto yield NO: 205) EgDHAsyn1*; no further nucleotide changes made pY143EgDHAsyn1C 10  9A 52 contains the N-terminal domain 20EloDom1 ofEgDHAsyn1* (SEQ ID (EgDHAsyn1C20EloDom1) and NO: 206) does not includethe proline-rich linker or delta-4 desaturase domain pY149 EgDHAsyn1C 10 9B 55 contains the N-terminal domain 20EloDom2Linker of EgDHAsyn1* aswell as the (SEQ ID proline-rich linker, but does not NO: 207) containthe delta-4 desaturase domain; also contains 4 additional amino acids(i.e., SCRT) after the linker region pY150 IgD4* 11  9C 62 Isochrysisgalbana delta-4 (SEQ ID desaturase with SbfI site at the NO: 210) 5′ endafter the start codon pY156 EgDHAsyn1C 11  9D 64 contains in-framefusion 20EloDom3- between the IgD4 EgDHAsyn1C20EloDom3Linker (“IgFus”)(SEQ and IgD4*, separated by the ID NO: 211) proline-rich linker regionpY152 EgDHAsyn1D 11 10A 67 contains the C-terminal domain 4Dom1 ofEgDHAsyn1* (i.e., the delta-4 (“EgD4Dom1”) desaturase domain, startingjust (SEQ ID after the end of the proline-rich NO: 216) linker region);also contains an ATG start codon at the 5′ end of the PCR productfollowed by an SbfI site pY157 EgDHAsyn1C 11 10B 69 contains an in-framefusion 20EloDom3- between EgD4Dom1 EgDHAsyn1C20EloDom and (“EgFus”)EgDHAsyn1D4Dom1, separated (SEQ ID by the proline-rich linker region NO:218) (called EgDHAsyn1C20EloDom3- EgD4Dom1); almost identical toEgDHAsyn1 except one amino acid (i.e., G323L) is changed due to the SbfIcloning site and fusion junction pY153 EgDHAsyn1D 11 10C 72 containsregion of the C- 4Dom2 (SEQ terminus of EgDHAsyn1 ID NO: 220) containingthe delta-4 “EgD4Dom2” desaturase domain and some of the C20 elongasedomain pY151 SaD4* 11 10D 76 Schizochytrium aggregatum (SEQ ID delta-4desaturase with SbfI site NO: 223) at the 5′ end after the start codonpY160 EgDHAsyn1C 11 11 77 contains in-frame fusion 20EloDom3- betweenSaD4 EgDHAsyn1C20EloDom3 and (“SaFus”) SaD4, separated by the proline-(SEQ ID rich linker region NO: 225)Fatty acid profiles of the transformant cells were subsequently analyzedas described in Example 27.

The results for feeding EPA to a vector only control, pY141, pY143,pY149, pY156, pY157 and pY160 are shown in FIG. 19. The fatty acidprofiles for Yarrowia expressing pY150, pY151, pY152 and pY153 showed noelongation of EPA to DPA and are not shown in FIG. 19.

The results for feeding DPA to a vector only control, pY141, pY150,pY151, pY152, pY153, pY156, pY157 and pY160 are shown in FIG. 20. Thefatty acid profiles for Yarrowia expressing pY143 and pY149 showed nodesaturation of DPA to DHA and are not shown in FIG. 20.

Percent C20 elongation (% C20 Elong), percent delta-4 desaturation (% D4Desat) and averages were calculated as described in Example 27.

In summary of FIG. 19 and FIG. 20, when the EgDHAsyn1C20Elo domain isexpressed alone (with or without the linker; i.e., pY143 and pY149), theaverage percent C20 elongation increases by about 40% compared to thenative EgDHAsyn1* (pY141; SEQ ID NO:49). The opposite occurs with theEgDHAsyn1 delta-4 desaturase domain where there is no activity withEgDHAsyn1D4Dom1 (pY152; SEQ ID NO:67; see FIG. 20) and about 50% lesswith EgDHAsyn1D4Dom2 (pY153; SEQ ID NO:72; see FIG. 20) when expressedalone compared to EgDHAsyn1* (pY141; SEQ ID NO:49; see FIG. 20) fed DPA.The IgD4 has no delta-4 desaturase activity when expressed alone (pY150;SEQ ID NO:62; see FIG. 20) or as a fusion (pY156; SEQ ID NO:64; seeFIGS. 19 and 20) and even causes an approximately 50% decrease inelongation activity when fused to the EgDHAsyn1C20 elongase domain(pY156; SEQ ID NO:64; see FIG. 19), possibly due to incorrect folding.In contrast, the SaD4 expressed alone (pY151; SEQ ID NO:76; see FIG. 20)has approximately the same delta-4 desaturase activity as that for thenative EgDHAsyn1* (pY141; SEQ ID NO:49; see FIG. 20). Interestingly, thedelta-4 desaturase activity of SaD4 increases approximately 2-fold whenfused to the EgDHAsyn1 C20 elongase domain (pY160; SEQ ID NO:77; seeFIG. 20). When fused to EgDHAsyn1 C20 elongase domain and fed EPA(pY160; SEQ ID NO:77; see FIG. 19), the delta-4 desaturase activity isapproximately 3-fold higher than when DPA is fed (pY160; SEQ ID NO:77;see FIG. 20) suggesting the linking of the two domains results inincreased efficiency or flux, perhaps due to substrate channeling.

Example 29 Substrate Specificity of EgDHAsyn1*

GLA, STA, EDA, ERA, DGLA, ETA, ARA, EPA and DPA were fed to Yarrowiacells transformed with pY141 (EgDHAsyn1*; SEQ ID NO:49) and a vectoronly control and fatty acid profiles were analyzed as described inExample 27.

The results for feeding EPA, ARA and DPA are shown in FIG. 22. The fattyacid profiles for Yarrowia fed with GLA, STA, EDA, ERA, DGLA and ETAshowed no elongation and are not shown in FIG. 22. Percent C20elongation (% C20 Elong) and percent delta-4 desaturation (% D4 Desat)and averages were calculated as described in Example 27 when fed EPA orDPA. When fed ARA, percent C20 elongation (% C20 Elong) was calculatedby dividing the sum of the wt. % for docosatetraenoic acid [DTA; 22:4(7,10,13,16)] and omega-6 docosapentaenoic acid [DPAn-6;22:5(4,7,10,13,16)] by the sum of the wt. % for ARA, DTA and DPAn-6 andmultiplying by 100 to express as a %. Similarly, percent delta-4desaturation (% D4 Desat) when fed ARA was calculated by dividing thewt. % for DPAn-6 by the sum of the wt. % for DTA and DPAn-6 andmultiplying by 100 to express as a %.

In summary of FIG. 22, EgDHAsyn1* elongates both ARA and EPA although ithas a slight preference (approximately 40% more active) for EPA. Theelongation product of ARA (i.e., DTA) is also desaturated in the delta-4position by EgDHAsyn1 to produce DPAn-6 and the activity isapproximately 40% higher for DTA than DPA.

Example 30 Co-expression of the Euglena gracilis DHA Synthase 1 with thePavlova lutheri Delta-8 Desaturase, the Mortierella alpina Delta-5Desaturase, the Saprolegnia diclina Delta-17 Desaturase and the Euglenagracilis Delta-9 Elongase in Soybean Embryos Transformed with SoybeanExpression Vectors pKR973 and pKR1064

Mature somatic soybean embryos are a good model for zygotic embryos.While in the globular embryo state in liquid culture, somatic soybeanembryos contain very low amounts of triacylglycerol or storage proteinstypical of maturing, zygotic soybean embryos. At this developmentalstage, the ratio of total triacylglyceride to total polar lipid(phospholipids and glycolipid) is about 1:4, as is typical of zygoticsoybean embryos at the developmental stage from which the somatic embryoculture was initiated. At the globular stage as well, the mRNAs for theprominent seed proteins, α′-subunit of β-conglycinin, kunitz trypsininhibitor 3, and seed lectin are essentially absent. Upon transfer tohormone-free media to allow differentiation to the maturing somaticembryo state, triacylglycerol becomes the most abundant lipid class. Aswell, mRNAs for α′-subunit of β-conglycinin, kunitz trypsin inhibitor 3and seed lectin become very abundant messages in the total mRNApopulation. On this basis, the somatic soybean embryo system behavesvery similarly to maturing zygotic soybean embryos in vivo, and is thusa good and rapid model system for analyzing the phenotypic effects ofmodifying the expression of genes in the fatty acid biosynthesis pathway(see PCT Publication No. WO 2002/00904, Example 3). Most importantly,the model system is also predictive of the fatty acid composition ofseeds from plants derived from transgenic embryos.

Fatty Acid Analysis of Transgenic Somatic Soybean Embryos ExpressingpKR973 and pKR1064:

Soybean embryogenic suspension cultures (cv. Jack) were transformed withthe AscI fragments of pKR973 and pKR1064 (fragments containing theexpression cassettes), as described for production in Example 25 and assummarized in Table 26.

TABLE 26 Summary of Vectors Expressed in Soybean Ex. FIG. SEQ ID VectorNo. No. NO: Genes Expressed pKR973 16 15B 128 Pavlova lutheri delta-8desaturase, Mortierella alpina delta-5 desaturase and Euglena gracilisdelta-9 elongase pKR1064 17 15C 132 Euglena gracilis DHA synthase 1 andSaprolegnia diclina delta-17 desaturase

A subset of soybean embryos generated from each event (ten embryos perevent) were harvested and picked into glass GC vials and fatty acidmethyl esters were prepared by transesterification. Fortransesterification, 50 μL of trimethylsulfonium hydroxide (TMSH) and0.5 mL of hexane were added to the embryos in glass vials and incubatedfor 30 min at room temperature while shaking. Fatty acid methyl esters(5 μL injected from hexane layer) were separated and quantified using aHewlett-Packard 6890 Gas Chromatograph fitted with an Omegawax 320 fusedsilica capillary column (Cat. No. 24152, Supelco Inc.). The oventemperature was programmed to hold at 220° C. for 2.6 min, increase to240° C. at 20° C./min and then hold for an additional 2.4 min. Carriergas was supplied by a Whatman hydrogen generator. Retention times werecompared to those for methyl esters of standards commercially available(Nu-Chek Prep, Inc.). Events having good phenotype were re-analyzed byGC using identical conditions except the oven temperature held at 150°C. for 1 min and then increased to 240° C. at 5° C.

The fatty acid profiles for individual embryos from a representativeevent are shown in FIG. 23. Fatty acids are identified as 16:0(palmitate), 18:0 (stearic acid), 18:1 (oleic acid), 18:2(LA), GLA, 18:3(ALA), EDA, DGLA, ARA, ERA, JUN, EPA, 22:3(10,13,16) (docosatrienoicacid), DTA, DPA and DHA; and, fatty acid compositions listed in FIG. 23are expressed as a weight percent (wt. %) of total fatty acids.

The activity of EgDHAsyn1 is expressed as percent C20 elongation (% C20Elong) and/or percent delta-4 desaturation (% D4 Desat), calculatedaccording to the following formula: ([product]/[substrate+product])*100.

More specifically, the percent elongation for EPA is shown as % C20Elong, determined as: ([DPA+DHA]/[EPA+DPA+DHA])*100. The percent delta-4desaturation for DPA is shown as % D4 Desat, determined as([DHA]/[DPA+DHA])*100. Other fatty acids that may be elongated ordesaturated were not included in this calculation.

In addition to elongation and desaturation products for EPA and ARA, itappears that in soybean, DGLA is also elongated by the EgDHAsyn1 as asignificant amount of the fatty acid 22:3(10,13,16) was made. The fattyacid was identified as 22:3(10,13,16) because it was found to have amass for 22:3 by GC-MS and had an MS profile that agrees with that for22:3(10,13,16).

Example 31 Expression of the Euglena gracilis Delta-9 Elongase/Pavlovalutheri Delta-8 Desaturase Fusion (EgD9elo-EgDHAsyn1Link-PavD8) inSoybean Embryos Transformed With Soybean Expression Vectors KS373

Soybean embryogenic suspension culture (cv. Jack) was transformed withKS373 (SEQ ID NO:179; FIG. 17) and KS120 (which is described in PCTPublication No. WO 2004/071467 and the contents of which are herebyincorporated by reference) as described for the model system in Example25. KS120 contains the hygromycin selection. KS373, produced in Example23, enabled expression of a fusion protein comprising the Euglenagracilis delta-9 elongase and the Pavlova lutheri delta-8 desaturase,wherein the two domains were linked with Euglena gracilis DHA Synthase 1Linker (i.e., EgDHAsyn1Link).

The fatty acid profiles for five individual embryos from 31 events wereobtained as described in Example 30. Results from the five bestelongation events are shown in FIG. 24. Fatty acids are identified as16:0 (palmitate), 18:0 (stearic acid), 18:1 (oleic acid), 18:2 (LA),GLA, 18:3 (ALA), EDA, DGLA, ERA and ETA; and, fatty acid compositionslisted in FIG. 24 are expressed as a weight percent (wt. %) of totalfatty acids.

The activity of EgD9elo-EgDHAsyn1Link-PavD8 is expressed as percentdelta-9 elongation (% D9 Elong) and/or percent delta-8 desaturation (%D8 Desat), calculated according to the following formula:([product]/[substrate+product])*100.

More specifically, the percent delta-9 elongation is shown as % D9Elong, determined as:([EDA+ERA+DGLA+ETA]/[LA+ALA+EDA+ERA+DGLA+ETA])*100. The percent delta-8desaturation is shown as % D8 Desat, determined as([DGLA+ETA]/[EDA+ERA+DGLA+ETA])*100.

The best % D9 Elong event had an average elongation of 22.1% with anaverage % D8 Desat of 92.7%. Elongation is slightly lower than that seenwhen the delta-9 elongase is expressed alone in soybean embryos althoughthis might be due to the small numbers of events looked at. In contrast,desaturation is considerably higher when the PavD8 is fused with theEgD9elo and EgDHAsyn1Link than when the PavD8 is expressed alone insoybean embryos, reaching almost 100% conversion in some events. Thisenhanced conversion by the delta-8 desaturase might be due to increasedefficiency or flux, perhaps due to substrate channeling.

Example 32 Synthesis And Functional Analysis of a Codon-Optimized C20Elongase Gene

(EqC20ES), From Euglena gracilis in Yarrowia lipolytica The codon usageof the C20 elongase domain of EgDHAsyn1 (EgDHAsyn1C20EloDom1) of Euglenagracilis (i.e., corresponding to amino acids 1-303 of SEQ ID NO:12) wasoptimized for expression in Yarrowia lipolytica, in a manner similar tothat described in PCT Publication No. WO 2004/101753 and U.S. Pat. No.7,125,672. Specifically, a codon-optimized C20 elongase gene (designated“EgC20ES” and having the nucleotide sequence as set forth in SEQ IDNO:183 and the amino acid sequence as set forth in SEQ ID NO:184) wasdesigned, based on the coding sequence of the C20 elongase domain ofEgDHAsyn1 (SEQ ID NO:201), according to the Yarrowia codon usage pattern(PCT Publication No. WO 2004/101753), the consensus sequence around the‘ATG’ translation initiation codon, and the general rules of RNAstability (Guhaniyogi, G. and J. Brewer, Gene, 265(1-2):11-23 (2001)).In addition to the modification of the translation initiation site, 163by of the 909 by coding region were modified (17.9%) and 147 codons wereoptimized (48.5%). None of the modifications in the codon-optimized genechanged the amino acid sequence of the encoded protein (i.e., SEQ IDNO:184 is 100% identical in sequence to amino acids 1-303 of SEQ IDNO:12). The designed EgC20ES gene (SEQ ID NO:183) was synthesized byGenScript Corporation (Piscataway, N.J.) and cloned into pUC57 (GenBankAccession No. Y14837) to generate pEgC20ES (FIG. 51B; SEQ ID NO:185).

To analyze the function of the codon-optimized EgC20ES gene, plasmidpZuFmEgC20ES (FIG. 52A; SEQ ID NO:360) comprising a chimericFBAINm::EgC20ES::Pex20 gene was constructed. Plasmid pZuFmEgC20EScontained the following components:

TABLE 27 Components Of Plasmid pZuFmEgC20ES (SEQ ID NO: 360) RE SitesAnd Nucleotides Within SEQ ID Description Of Fragment And Chimeric GeneNO: 360 Components SwaI/BsiWI FBAINm::EgC20ES::Pex20, comprising:(6063-318) FBAINm: Yarrowia lipolytica FBAINm promoter (PCT PublicationNo. WO 2005/049805; U.S. Pat. No. 7,202,356); EgC20ES: codon-optimizedC20 elongase gene (SEQ ID NO: 183), derived from Euglena gracilis;Pex20: Pex20 terminator sequence of Yarrowia Pex20 gene (GenBankAccession No. AF054613) 2269-1389 ColE1 plasmid origin of replication3199-2339 Ampicillin-resistance gene (Amp^(R)) for selection in E. coli4098-5402 Yarrowia autonomous replication sequence (ARS18; GenBankAccession No. A17608) 6935-5448 Yarrowia Ura 3 gene (GenBank AccessionNo. AJ306421)

Plasmid pZuFmEgC20ES was transformed into Yarrowia lipolytica strainY4184U4, as described in the General Methods. The transformants wereselected on MM plates. After 2 days growth at 30° C., 10 transformantsgrown on the MM plates were picked and re-streaked onto fresh MM plates.Once grown, 10 strains were individually inoculated into 3 mL liquid MMat 30° C. and shaken at 250 rpm/min for 2 days. The cells were collectedby centrifugation; lipids were extracted; and fatty acid methyl esterswere prepared by trans-esterification and subsequently analyzed with aHewlett-Packard 6890 GC.

GC analyses showed that there were about 6.2% EPA and 2.8% DPA of totallipids produced in all ten transformants, wherein the conversionefficiency of EPA to DPA in these 10 strains was determined to be about31% (calculated as described in Example 27). Thus, this experimentaldata demonstrated that the synthetic Euglena gracilis C20 elongasecodon-optimized for expression in Yarrowia lipolytica (i.e., EgC20ES, asset forth in SEQ ID NO:183) actively converts EPA to DPA.

Example 33 Synthesis and Functional Analysis of a Codon-Optimized C20Elongase Gene (EaC20ES) From Euglena anabaena in Yarrowia lipolytica

The codon usage of the C20 elongase domain of EaDHAsyn2 (SEQ ID NO:228)of Euglena anabaena (i.e., corresponding to amino acids 1-299 of SEQ IDNO:96) was optimized for expression in Yarrowia lipolytica, in a mannersimilar to that described in PCT Publication No. WO 2004/101753, U.S.Pat. No. 7,125,672 and above in Example 32. Specifically, acodon-optimized C20 elongase gene (designated “EaC20ES” and having thenucleotide sequence as set forth in SEQ ID NO:188 and the amino acidsequence as set forth in SEQ ID NO:189) was designed, based on thecoding sequence of the C20 elongase domain of EaDHAsyn2 (SEQ ID NO:92),according to the Yarrowia codon usage pattern (PCT Publication No. WO2004/101753), the consensus sequence around the ‘ATG’ translationinitiation codon, and the general rules of RNA stability (Guhaniyogi, G.and J. Brewer, Gene, 265(1-2):11-23 (2001)). In addition to themodification of the translation initiation site, 143 by of the 897 bycoding region were modified (15.9%) and 134 codons were optimized(44.8%). None of the modifications in the codon-optimized gene changedthe amino acid sequence of the encoded protein (i.e., SEQ ID NO:189 is100% identical in sequence to amino acids 1-299 of SEQ ID NO:96). Thedesigned EaC20ES gene (SEQ ID NO:188) was synthesized by GenScriptCorporation (Piscataway, N.J.) and was cloned into pUC57 (GenBankAccession No. Y14837) to generate pEaC20ES (SEQ ID NO:190).

To analyze the function of the codon-optimized EaC20ES gene, plasmidpZuFmEaC20ES (SEQ ID NO:361) was constructed comprising a chimericFBAINm::EaC20ES::Pex20 gene. Plasmid pZuFmEaC20ES (SEQ ID NO: 361) wasidentical in construction to that of plasmid pZuFmEgC20ES (SEQ IDNO:360; FIG. 52A), with the exception that EaC20ES (SEQ ID NO:188) wasused in place of EgC20ES (SEQ ID NO:183).

Plasmid pZuFmEaC20ES (SEQ ID NO:361) was transformed into Yarrowialipolytica strain Y4184U4, as described in the General Methods. Thetransformants were selected on MM plates. After 2 days growth at 30° C.,20 transformants grown on the MM plates were picked and re-streaked ontofresh MM plates. Once grown, 20 strains were individually inoculatedinto 3 mL liquid MM at 30° C. and shaken at 250 rpm/min for 2 days. Thecells were collected by centrifugation; lipids were extracted; and fattyacid methyl esters were prepared by trans-esterification andsubsequently analyzed with a Hewlett-Packard 6890 GC.

GC analyses showed that there were about 7.4% EPA and 1% DPA of totallipids produced in all 20 transformants, wherein the conversionefficiency of EPA to DPA in these 20 strains was determined to be about11% (calculated as described in Example 27). Thus, this experimentaldata demonstrated that the synthetic Euglena anabaena C20 elongasecodon-optimized for expression in Yarrowia lipolytica (i.e., EaC20ES, asset forth in SEQ ID NO:188) actively converts EPA to DPA.

Example 34 Synthesis and Functional Analysis of a Codon-OptimizedDelta-4 Desaturase Gene (EaD4S) From Euglena anabaena in Yarrowialipolytica

The codon usage of the delta-4 desaturase domain of EaDHAsyn2 (SEQ IDNO:243) of Euglena anabaena (i.e., corresponding to amino acids 259-841of SEQ ID NO:96) was optimized for expression in Yarrowia lipolytica, ina manner similar to that described in PCT Publication No. WO2004/101753, U.S. Pat. No. 7,125,672 and above in Examples 32 and 33.Specifically, a codon-optimized delta-4 desaturase gene (designated“EaD4S” and having the nucleotide sequence as set forth in SEQ ID NO:192and the amino acid sequence as set forth in SEQ ID NO:193) was designed,based on the coding sequence of the delta-4 desaturase domain ofEaDHAsyn2 (SEQ ID NO:92), which is also provided as SEQ ID NO:194(nucleotide) and SEQ ID NO:195 (amino acid). In addition to themodification of the translation initiation site, 307 by of the 1752(including the TAA stop codon) by coding region were modified (17.5%)and 285 codons were optimized (48.8%). Additionally, a NcoI site wasintroduced around the translation start codon by changing the secondamino acid of the wild type delta-4 desaturase domain (i.e., amino acidresidue 260 of SEQ ID NO:96 or amino acid residue 2 of SEQ ID NO:195)from a leucine to a valine residue in the synthetic EaD4S gene; thus,the amino acid sequence of EaD4S is set forth in SEQ ID NO:193. Thedesigned EaD4S gene (SEQ ID NO:192) was synthesized by GenScriptCorporation (Piscataway, N.J.) and was cloned into pUC57 (GenBankAccession No. Y14837) to generate pEaD4S (SEQ ID NO:196).

To analyze the function of the codon-optimized EaD4S gene, plasmidpZKL4-220EA4 (FIG. 52B; SEQ ID NO:362) was constructed to integrate twochimeric C20 elongase genes and the chimeric EaD4S gene into the lipase4 like locus (GenBank Accession No. XM_(—)503825) of Yarrowia lipolyticastrain Y4184U4. Plasmid pZKL4-220EA4 contained the following components:

TABLE 28 Components Of Plasmid pZKL4-220EA4 (SEQ ID NO: 362) RE SitesAnd Nucleotides Within SEQ ID Description Of Fragment And Chimeric GeneNO: 362 Components Asc I/BsiW I 745 bp 5′ portion of the Yarrowia Lipase4 like gene (4875-4123) (SEQ ID NO: 363; GenBank Accession No.XM_503825) PacI/SphI 782 bp 3′ portion of Yarrowia Lipase 4 like gene(SEQ (8371-7583) ID NO: 363; GenBank Accession No. XM_503825) Swa I/BsiWI FBAINm::EaC20ES::Pex20, comprising: (1980-4123) FBAINm: Yarrowialipolytica FBAINm promoter (PCT Publication No. WO 2005/049805; U.S.Pat. No. 7,202,356); EaC20ES: codon-optimized C20 elongase gene (SEQ IDNO: 188; Example 33), derived from Euglena anabaena; Pex20: Pex20terminator sequence from Yarrowia Pex20 gene (GenBank Accession No.AF054613) Pme I/Swa I YAT1::EgC20ES::Lip1, comprising: (1-1980) YAT1:Yarrowia lipolytica YAT1 promoter (Patent Publication No. U.S.2006/0094102-A1); EgC20ES: codon-optimized C20 elongase gene (SEQ ID NO:183; Example 32), derived from Euglena gracilis; Lip1: Lip1 terminatorsequence from Yarrowia Lip1 gene (GenBank Accession No. Z50020) ClaI/Pme I EXP1::EaD4S::Lip2, comprising: (1-10472) EXP1: Yarrowialipolytica export protein promoter (PCT Publication No. WO 2006/052870and U.S. Patent Application No. 11/265,761); EaD4S: codon-optimizeddelta-4 desaturase gene (SEQ ID NO: 192), derived from Euglena anabaena;Lip2: Lip2 terminator sequence from Yarrowia Lip2 gene (GenBankAccession No. AJ012632) Sal I/EcoR I Yarrowia Ura3 gene (GenBankAccession No. (10022-8403) AJ306421

Plasmid pZKL4-220EA4 was digested with AscI/SphI, and then transformedinto Yarrowia lipolytica strain Y4184U4, as described in the GeneralMethods. The transformants were selected on MM plates. After 5 daysgrowth at 30° C., 8 transformants grown on the MM plates were picked andre-streaked onto fresh MM plates. Once grown, these strains wereindividually inoculated into 3 mL liquid MM at 30° C. and shaken at 250rpm/min for 2 days. The cells were collected by centrifugation,resuspended in HGM and then shaken at 250 rpm/min for 5 days. The cellswere collected by centrifugation, lipids were extracted, and fatty acidmethyl esters were prepared by trans-esterification, and subsequentlyanalyzed with a Hewlett-Packard 6890 GC.

GC analyses showed that there were an average of 0.4% DHA and 10.2% DPAof total lipids produced in all 8 transformants, wherein the conversionefficiency of DPA to DHA in these 8 strains was determined to be about4.2% (calculated as described in Example 27). Thus, this experimentaldata demonstrated that the synthetic Euglena anabaena delta-4 desaturasecodon-optimized for expression in Yarrowia lipolytica (i.e., EaD4S, asset forth in SEQ ID NO:192) is active, but functions with relatively lowconversion efficiency.

Example 35 Co-Expression of the Euglena gracilis DHA Synthase 1 C20Elongase Domain (EgDHAsyn1C20EloDom1) with the Schizochytrium aggregatumDelta-4 Desaturase (SaD4) in Soybean Embryos Transformed with SoybeanExpression Vector pKR1105

The following example describes the generation of transgenic soybeanevents expressing EgDHAsyn1C20EloDom1 and SaD4 that, when generated intoplants, could be crossed with EPA-producing soybean events to generateDHA-producing plants.

Soybean embryogenic suspension culture (cv. Jack) was transformed withthe AscI fragment of pKR1105 (SEQ ID NO:156; FIG. 16A; fragmentcontaining the expression cassette) and embryos were matured asdescribed for production in Example 25 but with the following change.After maturation on SB103 for 10-12 days, a single cluster of embryosfor each event was removed to 4 mL of SB148 liquid media (recipe below)containing 0.02% tergitol and 0.33 mM EPA in a six-well micro-titerplate.

SB 103 Solid Medium (Per Liter)

-   -   1 package MS salts (Gibco/BRL—Cat. No. 11117-066)    -   1 mL B5 vitamins 1000× stock    -   60 g maltose    -   pH 5.7

Clusters were carefully broken up to release individual embryos andmicro-titer plates were shaken on a rotary shaker at 150 rpm and 26° C.under cool white fluorescent lights on a 16:8 hr day/night photoperiodat a light intensity of 60-85 μE/m2/s for 48 hrs. After 48 hrs, embryoswere rinsed with water, dried and five embryos per event were pickedinto glass GC vials. Fatty acid methyl esters were prepared bytransesterification with TMSH and were quantified using aHewlett-Packard 6890 Gas Chromatograph fitted with an Omegawax 320 fusedsilica capillary column (Cat. No. 24152, Supelco Inc.) as described inExample 30. The oven temperature was programmed to hold at 150° C. for 1min and then was increased to 240° C. at 5° C. Retention times werecompared to those for methyl esters of standards commercially available(Nu-Chek Prep, Inc.).

In this way, 122 events transformed with pKR1105 were analyzed. From the122 events analyzed, 49 were identified that elongated EPA (C20/delta-5elongase activity) and of these, 41 were identified that desaturated DPA(delta-4 desaturase activity) to produce DHA. The events with the bestC20/delta-5 elongase and delta-4 desaturase activities were advanced andthe fatty acid profiles from feeding embryos with EPA are shown in FIG.26.

Fatty acids in FIG. 26 are identified as 16:0 (palmitate), 18:0 (stearicacid), 18:1 (oleic acid), LA, ALA, EPA, 22:0 (docosanoic acid), DPA,24:0 (tetracosanoic acid), DHA and 24:1 (nevonic acid); and, fatty acidcompositions listed in FIG. 26 are expressed as a weight percent (wt. %)of total fatty acids. The activity of the EgDHAsyn1C20EloDom1 isexpressed as percent C20/delta-5 elongation (% C20/delta-5 elong),calculated according to the following formula:([product]/[substrate+product])*100. More specifically, the combinedpercent elongation for EPA is shown as “% C20/delta-5 elong”, determinedas: ([DPA+DHA]/[EPA+DPA+DHA])*100.

The activity of the SaD4 is expressed as percent delta-4 desaturation (%delta-4 desat), calculated according to the following formula:([product]/[substrate+product])*100. More specifically, the combinedpercent desaturation for DPA is shown as “% delta-4 desat”, determinedas: (DHA/[DPA+DHA])*100.

Example 36 Identification of a Delta-9 Elongase From Euglena anabaenaUTEX 373

The present example describes the identification of delta-9 elongasesfrom a Euglena anabaena UTEX 373 cDNA library. This work is alsodescribed in U.S. Provisional Application No. 60/911,925 (filed Apr. 16,2007; Attorney Docket No. BB-1613; the contents of which are herebyincorporated by reference).

Growth of Euglena anabaena UTEX 373 and Preparation of RNA

Amplified cDNA library eug1c was plated and colonies lifted as describedin Example 13. A DNA probe was made using an agarose gel purifiedNcoI/NotI DNA fragment containing the Euglena gracilis delta-9 elongasegene, from pKR906 (SEQ ID NO:115; Example 16 and WO 2007/061845, whichpublished May 31, 2007; Attorney Docket No. BB-1562; the contents ofwhich are hereby incorporated by reference) labeled with P³² dCTP usingthe RadPrime DNA Labeling System (Cat. No. 18428-011, Invitrogen,Carlsbad, Calif.) following the manufacturer's instructions.

Colony lifts were probed and positives were identified and confirmed asdescribed in Example 13. Plasmid DNA was isolated and sequenced exactlyas described in Example 2 and sequences were aligned and compared usingSequencher™ (Version 4.2, Gene Codes Corporation, Ann Arbor, Mich.). Inthis way, the clones could be categorized into one of two distinctgroups based on insert sequence (designated EaD9Elo1 and EaD9Elo2).Representative clones containing the cDNA for each class of sequencewere chosen for further study, and the sequences for each representativeplasmid (pLF121-1 and pLF121-2) are shown in SEQ ID NO:250 and SEQ IDNO:251, respectively. The sequence shown by a string of NNNN'srepresents a region of the polyA tail which was not sequenced. Thecoding sequences for EaD9Elo1 and EaD9Elo2 are shown in SEQ ID NO:252and SEQ ID NO:253, respectively. The corresponding amino acid sequencesfor EaD9Elo1 and EaD9Elo2 are shown in SEQ ID NO:254 and SEQ ID NO:255,respectively.

Example 37 Identification of a Delta-5 Desaturase from Euglena anabaenaUTEX 373

The present Example describes the identification of a delta-5 desaturasefrom Euglena anabaena UTEX 373. This work is also described in U.S.Provisional Application No. 60/915,733 (filed May 3, 2007; AttorneyDocket No. BB-1614; the contents of which are hereby incorporated byreference).

Amplified cDNA library eug1c was plated and colonies lifted as describedin Example 13. A DNA probe was made using an agarose gel purifiedNcoI/NotI DNA fragment containing the Euglena gracilis delta-5desaturase gene (EgD5; SEQ ID NO:267) from pDMW367, previously describedin PCT Publication No. WO 2007/136877 (published Nov. 29, 2007; AttorneyDocket No. BB1629; the contents of which are hereby incorporated byreference), labeled with P³².

Colony lifts were probed and positives were identified and confirmed asdescribed in Example 13. Plasmid DNA was isolated and sequenced exactlyas described in Example 2, and sequences were aligned and compared usingSequencher™ (Version 4.2, Gene Codes Corporation, Ann Arbor, Mich.).

A representative clone containing a cDNA (pLF119) is shown in SEQ IDNO:256 and the gene contained within the cDNA was called EaD5Des1. Thecoding sequence for EaD5Des1 is shown in SEQ ID NO:257. Thecorresponding amino acid sequence for EaD5Des1 is shown in SEQ IDNO:258.

Example 38 Construction of Soybean Expression Vector pKR1183 forExpression of a Euglena anabaena delta-9 elongase-Tetruetreptiapomquetensis CCMP1491 Delta-8 Desaturase Fusion Gene (Hybrid1-HGLASynthase)

An in-frame fusion between the Euglena anabaena delta-9 elongase(EaD9Elo1; SEQ ID NO:252; Example 36), the Euglena gracilis DHA synthase1 proline-rich linker (EgDHAsyn1Link; SEQ ID NO:197; Example 6) and theTetruetreptia pomquetensis CCMP1491 delta-8 desaturase (TpomD8; SEQ IDNO:162; Example 21; see also Applicants' Assignee's co-pendingapplication having U.S. patent application Ser. No. 11/876,115 (filedOct. 22, 2007; Attorney Docket No. BB-1574)) was constructed using theconditions described below.

An initial in-frame fusion between the EaD9Elo1 and the EgDHAsyn1Link(EaD9elo-EgDHAsyn1Link) was made, flanked by an NcoI site at the 5′ endand a NotI site at the 3′ end, by PCR amplification. EaD9Elo1 (SEQ IDNO:252) was amplified from pLF121-1 (SEQ ID NO:250) witholigonucleotides EaD9-5Bbs (SEQ ID NO:259) and EaD9-3fusion (SEQ IDNO:260), using the Phusion™ High-Fidelity DNA Polymerase (Cat. No.F553S, Finnzymes Oy, Finland) following the manufacturer's protocol.EgDHAsyn1Link (SEQ ID NO:197) was amplified in a similar way frompKR1049 (Example 4) with oligonucleotides EgDHAsyn1Link-5fusion (SEQ IDNO:261) and MWG511 (SEQ ID NO:175). The two resulting PCR products werecombined and re-amplified using EaD9-5Bbs (SEQ ID NO:259) and MWG511(SEQ ID NO:175) to form EaD9Elo1-EgDHAsyn1Link. The sequence of theEaD9Elo1-EgDHAsyn1Link is shown in SEQ ID NO:262. EaD9Elo1-EgDHAsyn1Linkdoes not contain an in-frame stop codon upstream of the NotI site at the3′ end and therefore, a DNA fragment cloned into the NotI site can giverise to an in-frame fusion with the EgD9elo1-EgDHAsyn1Link if thecorrect frame is chosen. EaD9Elo1-EgDHAsyn1Link was cloned into thepCR-Blunt® cloning vector using the Zero Blunt® PCR Cloning Kit(Invitrogen Corporation), following the manufacturer's protocol, toproduce pLF124 (SEQ ID NO:263).

The BbsI/NotI DNA fragment of pLF124 (SEQ ID NO:263), containingEaD9Elo1-EgDHAsyn1Link, was cloned into the NcoI/NotI DNA fragment fromKS366 (SEQ ID NO:177; Example 23), containing the promoter for the α′subunit of β-conglycinin, to produce pKR1177 (SEQ ID NO:264).

The BamHI DNA fragment of pKR1177 (SEQ ID NO:264), containingEaD9Elo1-EgDHAsyn1Link, was cloned into the BamHI DNA fragment ofpKR325, previously described in PCT Publication No. WO 2006/012325 (thecontents of which are hereby incorporated by reference) to producepKR1179 (SEQ ID NO:265).

The NotI fragment from pLF114-10 (Example 21; SEQ ID NO:165), containingTpomD8 was cloned into the NotI fragment of pKR1179 (SEQ ID NO:265) toproduce pKR1183 (SEQ ID NO:266; FIG. 28). In FIG. 28, the fusion gene(Hybrid1-HGLA synthase) is called EAd9ELONG-TPOMd8DS.

Example 39 Construction of Soybean Expression Vector pKR1253 forExpression of a Euglena anabaena Delta-9 Elongase-Tetruetreptiapomguetensis CCMP1491 Delta-8 Desaturase Fusion Gene (Hybrid1-HGLASynthase) with a Euglena gracilis Delta-5 Desaturase

Through a number of subcloning steps, a NotI site was added to the 5′end of the Euglena gracilis delta-5 desaturase (EgD5; SEQ ID NO:267)from pDMW367 and this NotI fragment containing EgD5 was cloned into theNotI site of pKR457 (SEQ ID NO:122; Example 16) to produce pKR1237 (SEQID NO:268).

The AscI fragment of pKR1183 (SEQ ID NO:266; Example 38), containing theHybrid1-HGLA synthase, was cloned into the AscI fragment of pKR277 (SEQID NO:120, which was previously described in PCT Publication No. WO2004/071467 and published Aug. 26, 2004; Attorney Docket No. BB-1538(the contents of which are hereby incorporated by reference) to producepKR1252 (SEQ ID NO:269).

The BsiWI fragment of pKR1237 (SEQ ID NO:268), containing the EgD5 gene,was cloned into the BsiWI site of pKR1252 (SEQ ID NO:269) to producepKR1253 (SEQ ID NO:270; FIG. 30).

Example 40 Construction of Soybean Vector pKR1139 for Expression of aEuglena anabaena Delta-5 Desaturase

The present example describes the cloning of a delta-5 desaturase fromEuglena anabaena UTEX 373 into a soybean expression vector. This work isalso described in U.S. Provisional Application No. 60/915,733 (filed May3, 2007; Attorney Docket No. BB-1614 (the contents of which are herebyincorporated by reference)).

EaD5Des1 (SEQ ID NO:257) was amplified from pLF119 (SEQ ID NO:256,Example 37) with oEAd5-1-1 (SEQ ID NO:271) and oEAd5-1-2 (SEQ IDNO:272), using the Phusion™ High-Fidelity DNA Polymerase (Cat. No.F553S, Finnzymes Oy, Finland) following the manufacturer's protocol. Theresulting PCR product was cloned into the pCR-Blunt® cloning vectorusing the Zero Blunt® PCR Cloning Kit (Invitrogen Corporation),following the manufacturer's protocol, to produce pKR1136 (SEQ IDNO:273).

The NotI fragment for pKR1136 (SEQ ID NO:273) containing the EaD5Des1was cloned into the NotI fragment of pKR974, previously described in PCTPublication No. WO 2007/136877, published Nov. 29, 2007 (Attorney DocketNo. BB-1629 (the contents of which are hereby incorporated byreference)), to produce pKR1139 (SEQ ID NO:274).

Example 41

Construction of Soybean Expression Vector pKR1255 for Expression of aEuglena anabaena Delta-9 Elongase-Tetruetreptia pomquetensis CCMP1491Delta-8 Desaturase Fusion Gene (Hybrid1-HGLA Synthase) with a Euglenagracilis Delta-5 Desaturase and a Euglena anabaena Delta-5 Desaturase

Plasmid pKR1139 (SEQ ID NO:274; Example 40) was digested with SbfI andthe fragment containing the EaD5Des1 was cloned into the SbfI site ofpKR1253 (SEQ ID NO:270; Example 39) to produce pKR1255 (SEQ ID NO:275;FIG. 31).

Example 42 Construction of Soybean Expression Vector pKR1189ForDown-Regulating Expression of Soybean Fad3

The present example describes a soybean expression vector designed todecrease fad3 expression in soybean.

A starting vector pKR561 (SEQ ID NO:276) was assembled by inserting theBsiWI fragment of pKR268 (previously described in PCT Publication No. WO04/071467) containing the annexin promoter into the BsiWI site ofpKR145, which is described in PCT Publication No. WO 04/071467.

Plasmid XF1, described in PCT Publication No. WO 93/11245 (which waspublished on Jun. 10, 1993; also U.S. Pat. No. 5,952,544; the contentsof which are hereby incorporated by reference), contains the soybeandelta-15 desaturase (fad3) gene (SEQ ID NO:277; GenBank Accession No.L22964; also called GmFAD3B).

A portion of the 5′ end of the fad3 gene was amplified from XF1 with thePhusion™ High-Fidelity DNA Polymerase (Cat. No. F553S, Finnzymes Oy,Finland) following the manufacturer's protocol, using HPfad3-1 (SEQ IDNO:278) and HPfad3-2 (SEQ ID NO:279) to produce a DNA fragment calledHPfad3AB (SEQ ID NO:280).

A portion of the 3′ end of the fad3 gene was amplified from XF1 with thePhusion™ High-Fidelity DNA Polymerase, using HPfad3-3 (SEQ ID NO:281)and HPfad3-1 (SEQ ID NO:278) to produce a DNA fragment called HPfad3A′-2(SEQ ID NO:282).

HPfad3AB and HPfad3A′-2 were combined and amplified using the Phusion™High-Fidelity DNA Polymerase with HPfad3-1 (SEQ ID NO:278) to produceHPfad3ABA′-2 (SEQ ID NO:283). HPfad3ABA′-2 (SEQ ID NO:283) has a NotIsite at both the 5′ and 3′ end of the DNA fragment. The resulting PCRproduct was cloned into the pCR-Blunt® cloning vector using the ZeroBlunt® PCR Cloning Kit (Invitrogen Corporation), following themanufacturer's protocol, to produce pLF129 (SEQ ID NO:284).

The NotI fragment for pLF129 (SEQ ID NO:284) containing the fad3 hairpinwas cloned into the NotI fragment of pKR561 (SEQ ID NO:276) to producepKR1189 (SEQ ID NO:285; FIG. 32). In FIG. 32, the A and A′ domains forfad3 are indicated by the designation TR1 while the B domain isindicated by TR2.

Example 43 Construction of Soybean Expression Vector pKR1249ForDown-Regulating Soybean Fad3 and Soybean Fad3c

The NotI/HindIII fragment of pLF129 (SEQ ID NO:284) containing the TR1and TR2 domains of fad3, as indicated in FIG. 32, was cloned into theNotI/HindIII backbone fragment of pLF129 (SEQ ID NO:284) to producepKR1209 (SEQ ID NO:286).

The coding sequence of GmFad3C (GenBank Accession No. AY204712) (Bilyeuet al., Crop Sci. 43:1833-1838 (2003); Anai et al., Plant Sci.168:1615-1623 (2005)) is shown in SEQ ID NO:287 and the correspondingamino acid sequence is shown in SEQ ID NO:288. A portion of the fad3cgene was amplified from the soybean cDNA library described in PCTPublication No. WO 93/11245 (which was published on Jun. 10, 1993; alsoU.S. Pat. No. 5,952,544) (the contents of which are hereby incorporatedby reference) with the Phusion™ High-Fidelity DNA Polymerase (Cat. No.F553S, Finnzymes Oy, Finland) following the manufacturer's protocol,using fad3c-5 (SEQ ID NO:289) and fad3c-3 (SEQ ID NO:290). The resultingDNA fragment was cloned into the pCR-Blunt® cloning vector using theZero Blunt® PCR Cloning Kit (Invitrogen Corporation), following themanufacturer's protocol, to produce pKR1213 (SEQ ID NO:291).

The EcoRV/XhoI fragment of pKR1213 (SEQ ID NO:291) containing thefragment of fad3c was cloned into the NotI(filled)/XhoI site of pKR1209(SEQ ID NO:286) to produce pKR1218 (SEQ ID NO:292).

The NotI/HindIII fragment of pLF129 (SEQ ID NO:284) containing the TR1domain only from fad3, as indicated in FIG. 32, was cloned into theNotI/HindIII backbone fragment of pLF129 (SEQ ID NO:284) to producepKR1210 (SEQ ID NO:293).

The EcoRV/XhoI fragment of pKR1213 (SEQ ID NO:291) containing thefragment of fad3c was cloned into the NotI(filled)/XhoI site of pKR1210(SEQ ID NO:293) to produce pKR1219 (SEQ ID NO:294).

The XhoI(filled)/HindIII fragment of pKR1218 (SEQ ID NO:292) containingthe fragment of fad3c as well as fad3 TR1 and TR2 domains was clonedinto the MluI (filled)/HindIII site of pKR1219 (SEQ ID NO:294),containing the fragment of fad3c as well as the fad3 TR1 only domain, toproduce pKR1225 (SEQ ID NO:295). In this way, a new hairpin includingfad3 and fad3c and flanked by NotI sites was formed.

The NotI fragment for pKR1225 (SEQ ID NO:295) containing the new hairpinincluding fad3 and fad3c was cloned into the NotI fragment of pKR561(SEQ ID NO:276; Example 42) to produce pKR1229 (SEQ ID NO:296; FIG. 33).In this way, the fad3/fad3c hairpin can be expressed from a strong,seed-specific promoter with hygromycin selection in plants.

The BsiWI fragment for pKR1225 (SEQ ID NO:295) containing the newhairpin including fad3 and fad3c was cloned into the BsiWI fragment ofpKR226 (SEQ ID NO:130; Example 17) to produce pKR1249 (SEQ ID NO:297;FIG. 34). In FIG. 34, pKR1249 is labeled pKR1249_PHP33240. In this way,the fad3/fad3c hairpin can be expressed from a strong, seed-specificpromoter with chlorsulfuron (ALS) selection in plants.

Example 44 Construction of Soybean Expression Vector pKR1322 forExpression of a Euglena anabaena Delta-9 Elongase-Tetruetreptiapomquetensis CCMP1491 Delta-8 Desaturase-Euglena anabaena Delta-5Desaturase Fusion Gene (EaD9Elo1-TpomD8-EaD5Des1 fusion)

The present example describes the construction of an in-frame fusiongene between the Euglena anabaena delta-9 elongase (EaD9Elo1; SEQ IDNO:252, Example 36), the Tetruetreptia pomquetensis CCMP1491 delta-8Desaturase (TpomD8; SEQ ID NO:162; Example 21) and the Euglena anabaenadelta-5 desaturase (EaD5Des1; SEQ ID NO:257; Example 37). Each domain isseparated by the EgDHAsyn1 linker (EgDHAsyn1Link; SEQ ID NO:197; Example6).

The EaD9Elo1-EgDHAsyn1Link (SEQ ID NO:262; Example 38) was amplifiedfrom pLF124 (SEQ ID NO:263) with oligonucleotides oEAd9el1-1 (SEQ IDNO:298) and oLINK-1 (SEQ ID NO:299), using the Phusion™ High-FidelityDNA Polymerase (Cat. No. F553S, Finnzymes Oy, Finland) following themanufacturer's protocol. EaD9Elo1-EgDHAsyn1Link is flanked by NotI atthe 5′ end and EagI at the 5′ and 3′ ends and does not contain anin-frame stop codon upstream of the EagI site at the 3′ end. Therefore,a DNA fragment cloned into the EagI site can give rise to an in-framefusion with the EgD9elo-EgDHAsyn1Link if the correct frame is chosen.The resulting DNA fragment containing EaD9Elo1-EgDHAsyn1Link was clonedinto the pCR-Blunt® cloning vector using the Zero Blunt® PCR Cloning Kit(Invitrogen Corporation), following the manufacturer's protocol, toproduce pKR1298 (SEQ ID NO:300).

An in-frame fusion between the TpomD8 and the EgDHAsyn1Link(TpomD8-EgDHAsyn1Link) was made which contained a NotI site at the 5′end and EagI sites at the 5′ and 3′ ends, by PCR amplification. TpomD8(SEQ ID NO:162) was amplified from pLF114-10 (SEQ ID NO:165) witholigonucleotides oTPd8-1 (SEQ ID NO:301) and oTPd8fus-1 (SEQ ID NO:302),using the Phusion™ High-Fidelity DNA Polymerase (Cat. No. F553S,Finnzymes Oy, Finland) following the manufacturer's protocol.EgDHAsyn1Link (SEQ ID NO:197) was amplified in a similar way frompKR1049 (Example 4) with oligonucleotides oLINK-2 (SEQ ID NO:303) andoLINK-1 (SEQ ID NO:304). The two resulting PCR products were combinedand re-amplified using oTPd8-1 (SEQ ID NO:301) and oLINK-1 (SEQ IDNO:304) to form TpomD8-EgDHAsyn1Link (SEQ ID NO:305).TpomD8-EgDHAsyn1Link was cloned into the pCR-Blunt® cloning vector usingthe Zero Blunt® PCR Cloning Kit (Invitrogen Corporation), following themanufacturer's protocol, to produce pKR1291 (SEQ ID NO:306).

The EagI fragment of pKR1291, containing the TpomD8-EgDHAsyn1Link wascloned into the NotI site of pBluescript II SK(+) vector (Stratagene) toform either pKR1301 (SEQ ID NO:307) in one orientation or pKR1301R (SEQID NO:308) in the opposite orientation.

Plasmid pKR1301 (SEQ ID NO:307) was digested with MfeI/BamHI, the DNAfragment containing TpomD8-EgDHAsyn1Link was completely filled in, andthe resulting DNA fragment was re-ligated to form pKR1311 (SEQ IDNO:309).

Plasmid pKR1301R (SEQ ID NO:308) was digested with EcoRI, and thefragment containing the 5′ end of TpomD8-EgDHAsyn1Link (calledTPOMD8TR2) and vector backbone was re-ligated to form pKR1304 (SEQ IDNO:310).

The EagI site of pKR1298 (SEQ ID NO:300) containing theEaD9Elo1-EgDHAsyn1Link was cloned into the EagI site of pKR1304 (SEQ IDNO:310) to produce pKR1309 (SEQ ID NO:311).

The NotI site of pKR1298 (SEQ ID NO:300) containing theEaD9Elo1-EgDHAsyn1Link was cloned into the EagI site of pKR1304 (SEQ IDNO:310) to produce pKR1309 (SEQ ID NO:311).

The NotI fragment for pKR1136 (SEQ ID NO:273; Example 40) containing theEaD5Des1 was cloned into the EagI site of pKR1311 (SEQ ID NO:309) toproduce pKR1313 (SEQ ID NO:312).

The MfeI/Ecl136II fragment of pKR1313 (SEQ ID NO:312) was cloned intothe EcoRV/MfeI sites of pKR1309 (SEQ ID NO:311) to produce pKR1315 (SEQID NO:313).

The NotI fragment of pKR1315 (SEQ ID NO:313) was cloned into the NotIsite of pKR72 (SEQ ID NO:105; Example 15) to produce pKR1322 (SEQ IDNO:314; FIG. 35).

In FIG. 35, the EaD9Elo1-TpomD8-EaD5Des1 fusion is labeled asEAd9el+TPd8ds+EAd5ds fusion.

Example 45 Down-Regulation of the Soybean Fad3 and fad3c Genes inSoybean Somatic Embryos by Transformation with pKR1189 or pKR1229

The present example describes the transformation and expression insoybean somatic embryos of pKR1189 (SEQ ID NO:285, Example 42),containing a fad3 hairpin construct or pKR1229 (SEQ ID NO:296; Example43), containing a fad3 and fad3c hairpin construct. Both constructs alsohave the hygromycin phosphotransferase gene for selection on hygromycin.

Soybean embryogenic suspension culture (cv. Jack) was transformed withpKR1189 (SEQ ID NO:285) or pKR1229 (SEQ ID NO:296) and embryos werematured in soybean histodifferentiation and maturation liquid medium(SHaM liquid media; Schmidt et al., Cell Biology and Morphogenesis,24:393 (2005)) as described in Example 25 and previously described inPCT Publication No. WO 2007/136877, published Nov. 29, 2007 (thecontents of which are hereby incorporated by reference).

After maturation in SHaM liquid media, individual embryos were removedfrom the clusters, dried and screened for alterations in their fattyacid compositions as described in Example 1. In each case, a subset ofsoybean embryos (i.e., five embryos per event) transformed with eitherpKR1189 (SEQ ID NO:285) or pKR1229 (SEQ ID NO:296) were harvested andanalyzed.

In this way, 41 events transformed with pKR1189 (SEQ ID NO:285;Experiment 2148) or pKR1229 (SEQ ID NO:296; Experiment 2165) wereanalyzed. The fatty acid profiles for the five events having the lowestaverage ALA content (average of the 5 embryos analyzed) along with anevent (2148-3-8-1) having a fatty acid profile typical of wild typeembryos for this experiment, are shown in FIG. 36. In FIG. 36, fattyacids are identified as 16:0 (palmitate), 18:0 (stearic acid), 18:1(oleic acid), LA, and ALA. Fatty acid compositions are expressed as aweight percent (wt. %) of total fatty acids.

ALA content in somatic embryos expressing either a fad3 hairpinconstruct (event number 2148, FIG. 36) or a fad3c hairpin construct(event number 2165, FIG. 36) showed at least a 50% reduction whencompared to typical wild type embryos (FIG. 36). This strongly indicatesthat either hairpin construct is functional to decrease ALA content insoybean embryos.

Example 46 Soybean Somatic Embryos Transformed with pKR1183 forExpression of a Euglena anabaena Delta-9 Elongase-Tetruetreptiapomquetensis CCMP1491 Delta-8 Desaturase Fusion Gene (Hybrid1-HGLASynthase)

The present example describes the transformation and expression insoybean somatic embryos of pKR1183 (SEQ ID NO:266; Example 38)containing the Euglena anabaena delta-9 elongase-Tetruetreptiapomquetensis CCMP1491 delta-8 Desaturase Fusion Gene (Hybrid1-HGLASynthase) and the hygromycin phosphotransferase gene for selection onhygromycin.

Soybean embryogenic suspension culture (cv. Jack) was transformed withpKR1183 (SEQ ID NO:266) and embryos were matured in soybeanhistodifferentiation and maturation liquid medium (SHaM liquid media;Schmidt et al., Cell Biology and Morphogenesis, 24:393 (2005)) asdescribed in Example 25 and previously described in PCT Publication No.WO 2007/136877, published Nov. 29, 2007 (the contents of which arehereby incorporated by reference).

After maturation in SHaM liquid media a subset of soybean embryos (i.e.,four embryos per event) transformed with pKR1183 (SEQ ID NO:266) wereharvested and analyzed as described herein.

In this way, 20 events transformed with pKR1183 (SEQ ID NO:266;Experiment 2145) were analyzed. The fatty acid profiles for the fiveevents having the highest average DGLA content (average of the 5 embryosanalyzed) are shown in FIG. 37. In FIG. 37, fatty acids are identifiedas 16:0 (palmitate), 18:0 (stearic acid), 18:1 (oleic acid), LA, ALA,EDA, ERA, DGLA and ETA. Fatty acid compositions are expressed as aweight percent (wt. %) of total fatty acids.

Example 47 Soybean Embryos Transformed with Soybean Expression VectorspKR1253 for Expression of a Euglena anabaena Delta-9Elongase-Tetruetreptia pomquetensis CCMP1491 Delta-8 Desaturase FusionGene (Hybrid1-HGLA Synthase) with a Euglena gracilis Delta-5 Desaturaseand pKR1249For Down-Regulating Soybean Fad3 and Soybean Fad3c

Soybean embryogenic suspension culture (cv. Jack) was transformed withthe AscI fragments of pKR1249 (SEQ ID NO:297; Example 43) and pKR1253(SEQ ID NO:270; Example 39) as described in Example 25. A subset ofsoybean embryos generated from each event (ten embryos per event) wereharvested, picked into glass GC vials and fatty acid methyl esters(FAMEs) were prepared by transesterification and analyzed by GC asdescribed in Example 1. Retention times were compared to those formethyl esters of standards commercially available (Nu-Chek Prep, Inc.).

In this way, 142 events transformed with pKR1249 (SEQ ID NO:297) andpKR1253 (SEQ ID NO:270) (experiment called Heal 25) were analyzed. Fromthe 142 events analyzed, 90 were identified that produced ARA in atleast one embryo out of ten analyzed at a relative abundance greaterthan 1.0% of the total fatty acids. Of these, 64 were identified thatproduced ARA in at least one embryo out of ten analyzed at a relativeabundance greater than 10.0% of the total fatty acids. Of these, 44events were identified that produced ARA in at least one embryo out often analyzed at a relative abundance greater than 20.0% of the totalfatty acids.

The average fatty acid profiles (Average of 10 embryos) for 20 eventshaving the highest ARA are shown in FIG. 38. Fatty acids are identifiedas 16:0 (palmitate), 18:0 (stearic acid), 18:1 (oleic acid), LA, ALA,EDA, SCI, DGLA, ARA, ERA, JUN, ETA and EPA; and fatty acid compositionslisted in FIG. 38 are expressed as a weight percent (wt. %) of totalfatty acids. For FIG. 38, fatty acids listed as “others” include: 18:2(5,9), 18:3 (5,9,12), STA, 20:0, 20:1(11), 20:2 (7,11) or 20:2 (8,11)and DPA. Each of these fatty acids is present at a relative abundance ofless than 2.0% of the total fatty acids. Average total omega-3 fattyacid (Total n-3) is the sum of the averages of all omega-3 fatty acids).

The actual fatty acid profiles for each embryo from one event (AFS5416-8-1-1) having an average ARA content of 17.0% and an average EPAcontent of 1.5% is shown in FIG. 39. Fatty acids are identified as 16:0(palmitate), 18:0 (stearic acid), 18:1 (oleic acid), LA, ALA, EDA, SCI,DGLA, ARA, ERA, JUN, ETA and EPA; and fatty acid compositions listed inFIG. 39 are expressed as a weight percent (wt. %) of total fatty acids.For FIG. 39, fatty acids listed as “others” include: 18:2 (5,9), 18:3(5,9,12), STA, 20:0, 20:1(11), 20:2 (7,11) or 20:2 (8,11) and DPA. Eachof these fatty acids is present at a relative abundance of less than2.0% of the total fatty acids. Total omega-3 fatty acid (Total n-3) isthe sum of all omega-3 fatty acids).

Because ALA contents is generally 1.5- to 3-fold higher in soybeansomatic embryos than it is in seed (i.e., 15%-30% in embryos (see, forexample, typical wild type embryo in FIG. 36), depending on maturationconditions and time versus 7-10% in a seed (Bilyeu et al., 2005, CropSci. 45:1830-1836), it is expected that omega-3 contents in general andEPA contents specifically, will be significantly lower in seed thansomatic embryos.

Example 48 Soybean Embryos Transformed with Soybean Expression VectorspKR1255 for Expression of a Euglena anabaena Delta-9Elongase-Tetruetreptia pomquetensis CCMP1491 Delta-8 Desaturase FusionGene (Hybrid1-HGLA Synthase) with a Euglena gracilis Delta-5 Desaturaseand a Euglena anabaena Delta-5 Desaturase and pKR1249For Down-RegulatingSoybean Fad3 and Soybean Fad3c

Soybean embryogenic suspension culture (cv. Jack) was transformed withthe AscI fragments of pKR1249 (SEQ ID NO:297; Example 43) and pKR1255(SEQ ID NO:275; Example 41) as described in Example 25. A subset ofsoybean embryos generated from each event (ten embryos per event) wereharvested, picked into glass GC vials and fatty acid methyl esters(FAMEs) were prepared by transesterification and analyzed by GC asdescribed in Example 1. Retention times were compared to those formethyl esters of standards commercially available (Nu-Chek Prep, Inc.).

In this way, 197 events transformed with pKR1249 (SEQ ID NO:297) andpKR1255 (SEQ ID NO:275) (experiment called Heal 26) were analyzed. Fromthe 197 events analyzed, 128 were identified that produced ARA in atleast one embryo out of ten analyzed at a relative abundance greaterthan 1.0% of the total fatty acids. Of these, 105 were identified thatproduced ARA in at least one embryo out of ten analyzed at a relativeabundance greater than 10.0% of the total fatty acids. And of these, 83events were identified that produced ARA in at least one embryo out often analyzed at a relative abundance greater than 20.0% of the totalfatty acids.

The average fatty acid profiles (Average of 9 or 10 embryos) for 20events having the highest ARA are shown in FIG. 40. Fatty acids areidentified as 16:0 (palmitate), 18:0 (stearic acid), 18:1 (oleic acid),LA, ALA, EDA, SCI, DGLA, ARA, ERA, JUN, ETA and EPA; and, fatty acidcompositions listed in FIG. 40 are expressed as a weight percent (wt. %)of total fatty acids. For FIG. 40, fatty acids listed as “others”include: 18:2 (5,9), 18:3 (5,9,12), STA, 20:0, 20:1(11), 20:2 (7,11) or20:2 (8,11) and DPA. Each of these fatty acids is present at a relativeabundance of less than 2.0% of the total fatty acids. Average totalomega-3 fatty acid (Total n-3) is the sum of the averages of all omega-3fatty acids).

Example 49 Expression of the Euglena gracilis DHA Synthase 1 C20Elongase Domain/Schizochytrium aggregatum Delta-4 Desaturase Fusion(EgDHAsyn1C20EloDom3-SaD4) Transformed with Soybean Expression VectorpKR1134

Soybean embryogenic suspension culture (cv. Jack) was transformed withthe AscI fragment of pKR1134 (SEQ ID NO:161; Example 20; fragmentcontaining the expression cassette) and embryos were matured asdescribed for production in Example 25. Substrate feeding of EPA andanalysis was carried out as described in Example 35.

In this way, 198 events transformed with pKR1134 (Experiment calledHeal24) were analyzed. From the 198 events analyzed, 193 were identifiedthat elongated EPA (C20/delta-5 elongase activity) and all of thesedesaturated DPA (delta-4 desaturase activity) to produce DHA to someextent. The events with the best C20/delta-5 elongase and delta-4desaturase activities were advanced and the fatty acid profiles fromfeeding embryos with EPA are shown in FIG. 41.

Fatty acids in FIG. 41 are identified as 16:0 (palmitate), 18:0 (stearicacid), 18:1 (oleic acid), LA, ALA, EPA, 22:0 (docosanoic acid), DPA,24:0 (tetracosanoic acid), DHA and 24:1 (nevonic acid); and fatty acidcompositions listed in FIG. 41 are expressed as a weight percent (wt. %)of total fatty acids. The activity of the EgDHAsyn1C20EloDom1 isexpressed as percent C20/delta-5 elongation (% C20/delta-5 elong),calculated according to the following formula:([product]/[substrate+product])*100. More specifically, the combinedpercent elongation for EPA is shown as “% C20/delta-5 elong”, determinedas: ([DPA+DHA]/[EPA+DPA+DHA])*100.

The activity of the SaD4 is expressed as percent delta-4 desaturation (%delta-4 desat), calculated according to the following formula:([product]/[substrate+product])*100. More specifically, the combinedpercent desaturation for DPA is shown as “% delta-4 desat”, determinedas: (DHA/[DPA+DHA])*100.

In addition to the 122 events analyzed for soy transformed with pKR1105(Experiment called Heal23) as described in Example 35, 20 more eventswere analyzed since the Example 35 was written bringing the totalanalyzed to 142 events. From the 20 new events analyzed, 18 wereidentified that elongated EPA (C20/delta-5 elongase activity) and 17 ofthese also desaturated DPA (delta-4 desaturase activity) to produce DHAto some extent. The events with the best C20/delta-5 elongase anddelta-4 desaturase activities from the 20 new events analyzed for soytransformed with pKR1105 were advanced and the fatty acid profiles fromfeeding embryos with EPA are shown in FIG. 42.

Relative activities of events transformed with either pKR1105 (C20elongase and delta-4 desaturase expressed individually) or pKR1134 (C20elongase and delta-4 desaturase expressed as a fusion) are compared byplotting % DHA (wt. %) vs. % DPA (wt. %) for all events where embryoswere fed EPA. The results are plotted in FIG. 43. In FIG. 43, Heal23 isthe name of the experiment where pKR1105 was transformed and Heal24 isthe experiment where pKR1134 was transformed. From FIG. 43, it is clearthat overall, DHA concentrations have increased while DPA concentrationshave decreased (to as low as undetectable), when the C20 elongase isfused to the delta-4 desaturase. Thus, fusing the 2 independent enzymestogether as one fusion protein separated by a linker region increasedflux from EPA to DHA.

Example 50 Expression of a Euglena anabaena Delta-9Elongase-Tetruetreptia pomquetensis CCMP1491 Delta-8 Desaturase-Euglenaanabaena delta-5 Desaturase Fusion Gene (EaD9Elo1-TpomD8-EaD5Des1fusion)

Soybean embryogenic suspension culture (cv. Jack) is transformed withpKR1322 (SEQ ID NO:314) and embryos are matured and analyzed for fattyacid profiles as described herein.

Soybean somatic embryos transformed with pKR1322 (SEQ ID NO:314) willelongate LA to EDA, EDA will be desaturated to DGLA, and DGLA will befurther desaturated to ARA. Because wild-type soybean also contains ALA,some ALA will be elongated to ERA, ERA will be desaturated to ETA, andETA will be further desaturated to EPA.

Soybean plants expressing the fusion gene from pKR1322 can beregenerated from embryos as described in Example 26 and seeds can beobtained.

In backgrounds that contain high ALA, or where a delta-15 desaturase andor delta-17 desaturase has been co-expressed, EPA will predominate.Conversely, ARA can be enriched by using a background low in ALA (forexample by crossing to a low ALA or low lin plant) or by knocking outthe endogenous fad3 gene(s) as described herein. Intermediate fattyacids (i.e., EDA and DGLA or ERA and ETA) will be lower when the fusionis used than when individual activities are transformed independently(i.e., not a fusion).

Other gene combinations (including linker combinations) can be fusedtogether in a similar way as described in Example 24. Similarly, otherpromoter/gene fusion/terminator combinations can be made.

Example 51 Determination of the Functional Domain in the SyntheticDelta-4 Desaturase Derived from Euglena anabaena and Codon-Optimized forExpression in Yarrowia lipolytica (EaD4S)

As schematically diagrammed in FIG. 52C, the C-terminal portion of theC20 elongase domain of EaDHAsyn1 (labeled as “EaC20E” in the figure)appears to overlap with the N-terminal portion of the delta-4 desaturasedomain of EaDHAsyn1 (labeled as “EaD4” in the figure). This is suggestedby sequence comparison.

In order to define the functional delta-4 desaturase domain in EaD4S(SEQ ID NO:192; Example 34), three EaD4S* mutants with differentN-terminal truncations were generated. Specifically, pZuFmEaD4S (SEQ IDNO:364) was constructed by replacing the NcoI/NotI fragment ofpZuFmIgD9ES (SEQ ID NO:365) with the NcoI/NotI EaD4S fragment of pEaD4S(SEQ ID NO:196; Example 34). A NcoI site was introduced into pZuFmEaD4S(SEQ ID NO:364) by site-directed mutagenesis using primer pairs YL921and YL922 (SEQ ID NOs:366 and 367, respectively), YL923 and YL924 (SEQID NOs:368 and 369, respectively) and YL925 and YL926 (SEQ ID NOs:370and 371, respectively) to generate pZuFmEaD4S-M1 (SEQ ID NO:372),pZuFmEaD4S-M2 (SEQ ID NO:373) and pZuFmEaD4S-M3 (SEQ ID NO:374),respectively. The pZuFmEaD4S-M1, pZuFmEaD4S-M2 and pZuFmEaD4S-M3plasmids were digested with NcoI, and then self-ligated to generatepZuFmEaD4S-1 (SEQ ID NO:375), pZuFmEaD4S-2 (SEQ ID NO:376) andpZuFmEaD4S-3 (SEQ ID NO:377) constructs. The NcoI/NotI fragmentscontaining different truncations of EaD4S from pZuFmEaD4S-1,pZuFmEaD4S-2, pZuFmEaD4S-3 were used to produce pZKL4-220EA4-1 (SEQ IDNO:378), pZKL4-220EA4-2 (SEQ ID NO:379) and pZKL4-220EA4-3 (SEQ IDNO:380) constructs. These three constructs were exactly the same aspZKL4-220EA4 (SEQ ID NO:362 and FIG. 52B, as described in Table 28 ofExample 34), except that the coding region of EaD4S was truncated at theN-terminal region. Specifically, instead of the 583 amino acid longcoding sequence of EaD4S (SEQ ID NO:193), the truncated EaD4S*polypeptide was 547 amino acids in length in pZKL4-220EA4-1 (i.e., SEQID NO:382), 527 amino acids in length in pZKL4-220EA4-2 (i.e., SEQ IDNO:384) and 512 amino acids in length in pZKL4-220EA4-3 (i.e., SEQ IDNO:386). The N-terminal region of these polypeptides (corresponding toamino acids 1-90 of SEQ ID NO:193) are aligned in FIG. 53A.

Plasmids pZKL4-220EA4 (SEQ ID NO:362), pZKL4-220EA4-1 (SEQ ID NO:378),pZKL4-220EA4-2 (SEQ ID NO:379) and pZKL4-220EA4-3 (SEQ ID NO:380) weredigested with AscI/SphI, and then transformed into Yarrowia lipolyticastrain Y4184U4, as described in the General Methods. Transformants wereselected on MM plates. After 5 days growth at 30° C., 5 transformantsgrown on the MM plates from each construct were picked and re-streakedonto fresh MM plates. Once grown, these strains were individuallyinoculated into 3 mL liquid MM at 30° C. and shaken at 250 rpm/min for 2days. The cells were collected by centrifugation, resuspended in HGM andthen shaken at 250 rpm/min for 5 days. The cells were collected bycentrifugation, lipids were extracted, and fatty acid methyl esters wereprepared by trans-esterification, and subsequently analyzed with aHewlett-Packard 6890 GC.

The GC results are shown below in Table 29. The composition of DPA andDHA are presented as a % of the total fatty acids. The conversionefficiency (“Conv. Effic.”) was measured according to the followingformula: ([DHA]/[DPA+DHA])*100. The delta-4 desaturase activity of eachtruncated EaD4S* (i.e., the polypeptides of 547 amino acids, 527 aminoacids or 512 amino acids in Yarrowia transformants of pZKL4-220EA4-1,pZKL4-220EA4-2 and pZKL4-220EA4-3, respectively) was compared to that ofEaD4S (i.e., Yarrowia transformants of pZKL4-220EA4) in the columnlabeled “% Delta-4 Activity”.

TABLE 29 Functional Analysis Of EaD4S And Truncated Variants In Yarrowialipolytica Strain Y4184U4 Delta-4 Desaturase Conv. % Plasmid Length DPADHA Effic. Delta-4 Transformant (SEQ ID NO) (%) (%) (%) ActivitypZKL4-220EA4 583 AA 10.2 0.4 4.2 100 (SEQ ID NO: 362) (SEQ ID NO: 193)pZKL4-220EA4-1 547 AA 8.0 2.2 21.5 512 (SEQ ID NO: 378) (SEQ ID NO: 382)pZKL4-220EA4-2 527 AA 11.5 1.8 13.2 314 (SEQ ID NO: 379) (SEQ ID NO:384) pZKL4-220EA4-3 512 AA 11.0 1.3 10.4 248 (SEQ ID NO: 380) (SEQ IDNO: 386)

These data demonstrated that the N-terminal 37 amino acids of EaD4S(i.e., amino acids 1-37 of SEQ ID NO:193) have a negative effect on theactivity of the delta-4 desaturase. Elevated delta-4 desaturase activityis measured with respect to EaD4S in each of the EaD4S* truncatedproteins, although the EaD4S* polypeptide of 547 amino acids (SEQ IDNO:382) is superior in activity as compared to the EaD4S* polypeptideslacking additional amino acids from their N-terminus (i.e., SEQ IDNOs:384 and 386).

Example 52 Synthesis and Functional Analysis of a Codon-OptimizedDelta-4 Desaturase Gene (EgD4S) From Euglena gracilis In Yarrowialipolytica

The codon usage of the delta-4 desaturase domain of EgDHAsyn1 (SEQ IDNO:221) of Euglena gracilis (corresponding to amino acids 253-793 of SEQID NO:12) was optimized for expression in Yarrowia lipolytica, in amanner similar to that described in PCT Publication No. WO 2004/101753,U.S. Pat. No. 7,125,672, and Examples 32, 33, and 34 herein.Specifically, a codon-optimized delta-4 desaturase gene (designated“EgD4S”; SEQ ID NO:387) was designed, based on the coding sequence ofthe delta-4 desaturase domain of EgDHAsyn1, according to the Yarrowiacodon usage pattern (PCT Publication No. WO 2004/101753), the consensussequence around the ‘ATG’ translation initiation codon, and the generalrules of RNA stability (Guhaniyogi, G. and J. Brewer, Gene,265(1-2):11-23 (2001)). In addition to the modification of thetranslation initiation site, 282 by of the 1623 by coding region weremodified (17.4%) and 270 codons were optimized (49.9%). Thecodon-optimized coding region of EgD4S is 1623 by in length, therebyencoding a polypeptide of 540 amino acids (SEQ ID NO:388). Thus, EgD4Sis one amino acid shorter in length than the wildtype delta-4 desaturasedomain of EgDHAsyn1 (i.e., SEQ ID NO:221; specifically, the leucineresidue corresponding to amino acid position 2 of SEQ ID NO:13 wasremoved in EgD4S (SEQ ID NO:388). The designed EgD4S gene (SEQ IDNO:387) was synthesized by GenScript Corporation (Piscataway, N.J.) andcloned into pUC57 (GenBank Accession No. Y14837) to generate pEgD4S (SEQID NO:389).

To analyze the function of the codon-optimized EgD4S gene, plasmidpZKL4-220Eg4 (SEQ ID NO:390) was constructed to integrate two chimericC20 elongase genes and the chimeric EgD4S gene into the lipase 4 likelocus (GenBank Accession No. XM_(—)503825) of Yarrowia lipolytica strainY4305U3. Plasmid pZKL4-220Eg4 (SEQ ID NO:390) was identical inconstruction to that of plasmid pZKL4-220Ea4 (SEQ ID NO:362; FIG. 52B;Table 28 of Example 34), with the exception that EgD4S (SEQ ID NO:387)was used in place of EaD4S (SEQ ID NO:192).

Plasmid pZKL4-220Eg4 was digested with AscI/SphI, and then transformedinto Yarrowia lipolytica strain Y4305U3, as described in the GeneralMethods. The transformants were selected on MM plates. After 5 daysgrowth at 30° C., 14 transformants grown on the MM plates were pickedand re-streaked onto fresh MM plates. Once grown, these strains wereindividually inoculated into 3 mL liquid MM at 30° C. and shaken at 250rpm/min for 2 days. The cells were collected by centrifugation,resuspended in HGM and then shaken at 250 rpm/min for 5 days. The cellswere collected by centrifugation, lipids were extracted, and fatty acidmethyl esters were prepared by trans-esterification, and subsequentlyanalyzed with a Hewlett-Packard 6890 GC.

GC analyses showed that there were an average of 4.9% DHA and 17.8% DPAof total lipids produced in all 14 transformants, wherein the conversionefficiency of DPA to DHA was determined to be about 21.5% (calculated asdescribed in Example 27). Thus, this experimental data demonstrated thatthe synthetic Euglena gracilis delta-4 desaturase codon-optimized forexpression in Yarrowia lipolytica (i.e., EgD4S, as set forth in SEQ IDNO:387) was active to convert DPA to DHA.

Example 53 Determination of the Functional Domain in the SyntheticDelta-4 Desaturase Derived from Euglena gracilis and Codon-Optimized forExpression in Yarrowia lipolytica (EgD4S)

In a manner similar to that observed in EaDHAsyn1 (FIG. 52C), theC-terminal portion of the C20 elongase domain of EgDHAsyn1 appears tooverlap with the N-terminal portion of the delta-4 desaturase domain ofEgDHAsyn1, based on sequence comparison.

In order to define the functional delta-4 desaturase domain in EgD4S(SEQ ID NO:387), three EgD4S mutants with different N-terminaltruncations were generated. A NcoI site was introduced into pEgD4S (SEQID NO:389; Example 52) by site-directed mutagenesis using primer pairsYL935 and YL936 (SEQ ID NOs:391 and 392, respectively), YL937 and YL938(SEQ ID NOs:393 and 394, respectively) and YL939 and YL940 (SEQ IDNOs:395 and 396, respectively) to generate pEgD4S-M1 (SEQ ID NO:397),pEgD4S-M2 (SEQ ID NO:398) and pEgD4S-M3 (SEQ ID NO:399) constructs,respectively. The NcoI/NotI fragments containing different truncationsof EgD4S from pEgD4S-M1, pEgD4S-M2 and pEgD4S-M3 were used to generatepZKL4-220Eg-4-1 (SEQ ID NO:400), pZKL4-220Eg-4-2 (SEQ ID NO:401) andpZKL4-EgD4-3 (SEQ ID NO:402) constructs. These three constructs wereexactly the same as pZKL4-220Eg4 (SEQ ID NO:390; Example 52), exceptthat the coding region of EgD4S was truncated at the N-terminal region.Specifically, instead of the 540 amino acid long coding sequence ofEgD4S (SEQ ID NO:388), the truncated EgD4S* polypeptide was 513 aminoacids in length in pZKL4-220Eg-4-1 (i.e., SEQ ID NO:404), 490 aminoacids in length in pZKL4-220Eg-4-2 (i.e., SEQ ID NO:406) and 474 aminoacids in length in pZKL4-220Eg-4-3 (i.e., SEQ ID NO:408). The N-terminalregion of these polypeptides (corresponding to amino acids 1-80 of SEQID NO:388) are aligned in FIG. 53B.

Plasmids pZKL4-220Eg4 (SEQ ID NO:390), pZKL4-220Eg-4-1 (SEQ ID NO:400),pZKL4-220Eg-4-2 (SEQ ID NO:401) and pZKL4-EgD4-3 (SEQ ID NO:402) weredigested with AscI/SphI, and then transformed into Yarrowia lipolyticastrain Y4305U3 individually, as described in the General Methods.Transformants were selected on MM plates. After 5 days growth at 30° C.,4 transformants grown on the MM plates from each construct were pickedand re-streaked onto fresh MM plates. Once grown, these strains wereindividually inoculated into 3 mL liquid MM at 30° C. and shaken at 250rpm/min for 2 days. The cells were collected by centrifugation,resuspended in HGM and then shaken at 250 rpm/min for 5 days. The cellswere collected by centrifugation, lipids were extracted, and fatty acidmethyl esters were prepared by trans-esterification, and subsequentlyanalyzed with a Hewlett-Packard 6890 GC.

The GC results are shown below in Table 30. The composition of DPA andDHA are presented as a % of the total fatty acids. The conversionefficiency (“Cony. Effic.”) was measured according to the followingformula: ([DHA]/[DPA+DHA])*100. The delta-4 desaturase activity of eachtruncated EgD4S* (i.e., the polypeptides of 513 amino acids, 490 aminoacids or 474 amino acids in Yarrowia transformants of pZKL4-220Eg-4-1,pZKL4-220Eg-4-2 and pZKL4-220Eg-4-3, respectively) was compared to thatof EgD4S (i.e., Yarrowia transformants of pZKL4-220Eg4) in the columnlabeled “% Delta-4 Activity”.

TABLE 30 Functional Analysis Of EgD4S And Truncated Variants In Yarrowialipolytica Strain Y4305U3 Delta-4 Desaturase Conv. % Plasmid Length DPADHA Effic. Delta-4 Transformant (SEQ ID NO) (%) (%) (%) ActivitypZKL4-220Eg4 540 AA 17.8 4.9 21.5 100 (SEQ ID NO: 390) (SEQ ID NO: 388)pZKL4-220Eg4-1 513 AA 16.5 4.6 21.6 100 (SEQ ID NO: 400) (SEQ ID NO:404) pZKL4-220Eg4-2 490 AA 16.8 3.8 18.5 86 (SEQ ID NO: 401) (SEQ ID NO:406) pZKL4-220Eg4-3 474 AA 24.7 2.4 8.8 41 (SEQ ID NO: 402) (SEQ ID NO:408)

These data demonstrated that the N-terminal 28 amino acids of EgD4S* aredispensable (i.e., see pZKL4-220Eg-4-1, where the first 28 amino acidsof EgD4S were truncated and the truncated protein set forth as SEQ IDNO:404 retained full delta-4 desaturase activity). Reduced delta-4desaturase activity was measured in transformants with pZKL4-220EgD4-2and pZKL4-220EgD4-3, when additional amino acids were truncated from the5′ portion of the EgD4S protein, thereby resulting in SEQ ID NO:406 andSEQ ID NO:408.

Example 54 Synthesis And Functional Analysis Of A Codon-OptimizedEgDHAsyn1 Gene (EgDHAsyn1S) From Euglena gracilis In Yarrowia lipolytica

Plasmid pZKLY-G204 (FIG. 54A; SEQ ID NO:409) was designed to integrate achimeric gene containing a codon-optimized EgDHAsyn1 coding region(i.e., EgDHAsyn1S, set forth as SEQ ID NOs:410 and 411) into the lipase7 locus (GenBank Accession No. AJ549519) of Yarrowia lipolytica strainY4305U3. In addition to the modification of the translation initiationsite, 417 by of the 2382 by coding region were modified (17.5%), and 391codons of the total 794 codons were optimized (49.2%). The amino acidsequence of the codon-optimized EgDHAsyn1S (SEQ ID NO:411) is 100%identical in sequence to that of EgDHAsyn1 (SEQ ID NO:12).

To generate pZKLY-G204, a KpnI site was introduced into pEgC20ES (SEQ IDNO:185; see FIG. 51B, Example 32) to generate pEgC20ES-K (SEQ ID NO:412;FIG. 54B) by site-directed mutagenesis using oligonucleotides YL973 andYL974 (SEQ ID NOs:413 and 414, respectively) as primers and pEgC20ES astemplate. A 732 by PmeI/NcoI fragment containing the YAT1 promoter(Patent Publication US 2006/0094102-A1) of pYNTGUS1-CNP (FIG. 54C; SEQID NO:415), the 873 by NcoI/KpnI fragment of pEgC20ES-K containing thecodon-optimized N-terminal portion of EgDHAsyn1S and the 1512 byKpnI/NcoI fragment of pEgD4S (SEQ ID NO:389; Example 52) containing thecodon-optimized C-terminal portion of EgDHAsyn1S were isolated, and thenused to replace the PmeI/NotI fragment of pZKLY (FIG. 54D; SEQ IDNO:416) to generate pZKLY-G204 (FIG. 54A). Thus, pZKLY-G204 containedthe following components:

TABLE 31 Components Of Plasmid pZKLY-G204 (SEQ ID NO: 409) RE Sites AndNucleotides Within SEQ ID Description Of Fragment And Chimeric Gene NO:409 Components Asc I/BsiW I 887 bp 5′ portion of the Yarrowia Lipase 7gene (SEQ (1383-489) ID NO: 417) (labeled as “LipY-5′” in Figure;GenBank Accession No. AJ549519) PacI/SphI 756 bp 3′ portion of YarrowiaLipase 7 gene (SEQ ID (4853-4091) NO: 417) (labeled as “LipY-3′” inFigure; GenBank Accession No. AJ549519) Pme I/BsiW IYAT1::EgDHAsyn1S::Lip1, comprising: (7301-333) YAT1: Yarrowia lipolyticaYAT1 promoter (Patent Publication No. US 2006/0094102-A1); EgDHAsyn1S:codon-optimized DHA synthase (SEQ ID NO: 410), derived from Euglenagracilis; Lip1: Lip1 terminator sequence from Yarrowia Lip1 gene(GenBank Accession No. Z50020) Sal I/EcoR I Yarrowia Ura3 gene (GenBankAccession No. (6504-4885) AJ306421)

The pZKLY-G204 plasmid was digested with AscI/SphI, and then transformedinto Yarrowia lipolytica strain Y4305U3, as described in the GeneralMethods. The transformants were selected on MM plates. After 5 daysgrowth at 30° C., 8 transformants grown on the MM plates were picked andre-streaked onto fresh MM plates. Once grown, these strains wereindividually inoculated into 3 mL liquid MM at 30° C. and shaken at 250rpm/min for 2 days. The cells were collected by centrifugation,resuspended in HGM and then shaken at 250 rpm/min for 5 days. The cellswere collected by centrifugation, lipids were extracted, and fatty acidmethyl esters were prepared by trans-esterification, and subsequentlyanalyzed with a Hewlett-Packard 6890 GC.

GC analyses showed that there were about 5.0% DHA, 13.5% DPA and 26.5%EPA of total lipids produced in all 8 transformants. The conversionefficiency of EPA to DPA and DHA was determined to be about 41%; and,the conversion efficiency of DPA to DHA was determined to be about 27%in these eight strains (calculated as described in Example 27). Thus,this experimental data demonstrated that the synthetic Euglena gracilisDHA synthase codon-optimized for expression in Yarrowia lipolytica(i.e., EgDHAsyn1S as set forth in SEQ ID NO:410) contained both C20elongase activity and delta-4 desaturase activity. EgDHAsyn1S could useEPA as substrate to produce DHA.

Example 55 Creation of Delta-9 Elongase/Delta-8 Desaturase Gene Fusionsfor Expression in Yarrowia lipolytica

In order to improve the enzyme activity of delta-9 elongase and delta-8desaturase in Yarrowia lipolytica, a series of six delta-9elongase/delta-8 gene fusions (multizymes) are created in the presentExample, using two variant linker sequences (i.e., SEQ ID NO:438[GPARPAGLPPATYYDSLAV] and SEQ ID NO:445 [GAGPARPAGLPPATYYDSLAVMGS])derived from the EgDHAsyn1 proline-rich linker (SEQ ID NO:198;PARPAGLPPATYYDSLAV).

This work required: identification of appropriate delta-9 elongases anddelta-8 desaturases for expression in Yarrowia lipolytica; and,construction of plasmid pZUFmG9G8fu (comprising a EgD9ES/EgD8M genefusion), plasmid pZuFmG9G8fu-B (comprising a EgD9ES/EgD8M gene fusion),plasmid pZUFmG9A8 (comprising a EgD9ES/EaD8S gene fusion), plasmidpZUFmA9G8 (comprising a EaD9ES/EgD8M gene fusion), plasmid pZUFmA9A8(comprising a EaD9ES/EaD8S gene fusion) and plasmid pZUFmR9G8(comprising a E389D9eS/EgD8M gene fusion). All plasmids shared a commonvector backbone and thus were distinguished only by the gene fusion thateach comprised.

Functional analysis of the activity of each gene fusion is tested infra,in Example 56.

Description of Synthetic Delta-9 Elongase and Delta-8 Desaturase GenesCodon-Optimized for Expression in Yarrowia lipolytica

The Applicants have performed considerable analyses of various delta-9elongases and delta-8 desaturases, to determine those enzymes havingoptimal substrate specificity and/or substrate selectivity whenexpressed in Yarrowia lipolytica. Based on these analyses, the genesdescribed below in Table 32, and codon-optimized genes derived therefrom (based on methodology of U.S. Pat. No. 7,125,672), were identifiedas preferred for expression in Y. lipolytica. Those genes highlighted inbold text were subsequently utilized to create delta-9 elongase/delta-8desaturase gene fusions.

TABLE 32 Preferred Desaturases And Elongases For Creation OfDelta-9/Delta-8 Gene Fusions In Yarrowia lipolytica WildtypeCodon-Optimized Mutant Co-pending Patent Application Abbreviation andAbbreviation and Abbreviation and ORF Organism References SEQ ID NOs SEQID NOs SEQ ID NO Delta-9 Euglena Patent Publication US 2007-0117190“EgD9e”* “EgD9eS” — Elongase gracilis A1; (SEQ ID NO: 112) (SEQ ID NOs:318 see also Example 16 herein and 319) Eutreptiella Patent PublicationUS 2007-0117190 “E389D9e” “E389D9eS” — sp. A1; (SEQ ID NOs: 419 (SEQ IDNOs: 358 CCMP389 and 420) and 359) Euglena Example 36 herein “EaD9e”*“EaD9eS” — anabaena (SEQ ID NOs: 252 (SEQ ID NOs: 421 UTEX 373 and 254)and 422) Delta-8 Euglena U.S. Pat. No. 7,256,033; “EgD8”* “EgD8S”*“EgD8M”* Desaturase gracilis U.S. Patent Application No. 11/635,258 (SEQID NOs: 423 (SEQ ID NOs: 425 (SEQ ID NOs: 327 and 424) and 426) and 328)Euglena “EaD8”* “EaD8S” — anabaena (SEQ ID NOs: 427 (SEQ ID NOs: 429UTEX 373 and 428) and 430) *Notes: EgD9e was described as “EgD9elo” inExample 16 herein. EaD9e was identified as “EaD9Elo1” in U.S.Provisional Patent Application No. 60/911,925 and in Example 36, herein.EgD8 was identified as “Eg5” in U.S. Pat. No. 7,256,033. EgD8S wasidentified as “D8SF” in U.S. Pat. No. 7,256,033. EgD8M was identified as“EgD8S-23” in U.S. Patent Application No. 11/635,258.

The LA to EDA conversion efficiency of EgD9eS, E389D9eS and EaD9eS isreported in each of the applications cited above. Briefly, however, wheneach delta-9 elongase was expressed as a chimeric gene in Yarrowialipolytica strain Y2224 (FIG. 44), under the control of a YarrowiaFBAINm promoter (PCT Publication No. WO 2005/049805; U.S. Pat. No.7,202,356) and a Pex20 terminator sequence from the Yarrowia Pex20 gene(GenBank Accession No. AF054613), the following substrate conversionswere independently measured: EgD9eS (20.1%); EaD9eS (13%); and, E389D9eS(12%). All synthetic codon-optimized genes functioned with greatersubstrate conversion efficiency than the corresponding wildtype gene.

U.S. Pat. No. 7,256,033 discloses a E. gracilis delta-8 desaturase(“EgD8”) able to desaturate EDA and EtrA to DGLA and ETA, respectively,as well as a synthetic delta-8 desaturase derived from EgD8 andcodon-optimized for expression in Yarrowia lipolytica (“EgD8S”). Despitethe usefulness of EgD8 and EgD8S, a synthetically engineered mutantdelta-8 desaturase identified herein as EgD8M (SEQ ID NOs:327 and 328)is used in more preferred embodiments for expression in Yarrowialipolytica. As described in U.S. patent application Ser. No. 11/635,258,“EgD8M” (identified therein as “EgD8S-23”) was created by makingmultiple rounds of targeted mutations within EgD8S. The effect of eachmutation on the delta-8 desaturase activity of the resulting mutant wasscreened to ensure functional equivalence with the delta-8 desaturaseactivity of EgD8S (SEQ ID NO:426). As a result of this work, mutantEgD8M (SEQ ID NO:328) comprises the following 24 amino acid mutationswith respect to the synthetic codon-optimized EgD8S sequence set forthas SEQ ID NO:426: 4S to A, 5K to S, 12T to V, 16T to K, 17T to V, 66P toQ, 67S to A, 108S to L, 117G to A, 118Y to F, 120L to M, 121 M to L,125Q to H, 126M to L, 132V to L, 133 L to V, 162L to V, 163V to L, 293Lto M, 407A to S, 408V to Q, 418A to G, 419G to A and 422L to Q. Pairwisealignment of the EgD8M and EgD8S protein sequences using defaultparameters of Vector NTI®'s AlignX program (Invitrogen Corporation,Carlsbad, Calif.) revealed 94.3% sequence identity and 97.9% consensusbetween the two proteins over a length of 422 amino acids. Average EDAto DGLA substrate conversion by this mutant delta-8 desaturase wasdetermined to be 37%, when EgD8M was expressed in Yarrowia lipolyticastrain Y4001 (FIG. 44), under the control of a Yarrowia FBAINm promoter(PCT Publication No. WO 2005/049805; U.S. Pat. No. 7,202,356) and aPex20 terminator sequence from the Yarrowia Pex20 gene (GenBankAccession No. AF054613).

When EaD8S was expressed in Yarrowia lipolytica strain Y4001U (FIG. 44),under the control of a Yarrowia FBAINm promoter U.S. Pat. No. 7,202,356)and a Pex20 terminator sequence from the Yarrowia Pex20 gene (GenBankAccession No. AF054613), there was 41% conversion efficiency to DGLAwith endogenous EDA as substrate.

Generation of Construct pZUFmEgD9ES-Na, Comprising EgD9ES

Plasmid pZuFmEgD9ES (SEQ ID NO:431), which was previously described inPatent Publication US 2007-0117190 A1, comprises a chimericFBAINm::EgD9ES::Pex20 gene, a ColE1 plasmid origin of replication, anampicillin-resistance gene (Amp^(R)) for selection in E. coli, aYarrowia autonomous replication sequence (ARS18; GenBank Accession No.A17608), and a Yarrowia Ura 3 gene (GenBank Accession No. AJ306421).

A Nar I site was introduced into pZuFmEgD9ES to generate pZuFmEgD9ES-Na(SEQ ID NO:432) using oligonucleotides YL989 and YL990 (SEQ ID NOs:433and 434, respectively) as primers and pZuFmEgD9ES as template. Theintroduced Nar I site (i.e., GGCGCC) was located just before thetranslation stop codon of EgD9ES; therefore, the coding region of EgD9ESwas extended with two additional amino acids (i.e., a glycine and analanine).

Generation of Construct pZUFmG9G8Fu, Comprising a EgD9ES/EgD8M GeneFusion

The N-terminal portion of EgD8M (SEQ ID NO:327) was amplified by PCRusing oligonucleotides YL991 and YL992 (SEQ ID NOs:435 and 436,respectively) as primers and pKO2UFm8A (SEQ ID NO:437) as template.Oligonucleotide YL991 contained a Nar I site at its 5′ end and DNAsequence encoding a modified variant of the EgDHAsyn1 proline-richlinker (i.e., GPARPAGLPPATYYDSLAV, as set forth in SEQ ID NO:438 versusPARPAGLPPATYYDSLAV, as set forth in SEQ ID NO:198). This linkerpossessed an additional glycine at the 5′ end, with respect to theEgDHAsyn1 proline-rich linker (SEQ ID NO:198).

The Nar I/Bgl II digested PCR product comprising the 5′ portion of EgD8Mand the Bgl II/Not I digested fragment of pKO2UFm8A comprising the 3′portion of EgD8M was used to replace the Nar I/Not I fragment ofpZUFmEgD9ES-Na to generate pZUFmG9G8fu (FIG. 55A), which therebycontained the following components:

TABLE 33 Components Of Plasmid pZUFmG9G8fu (SEQ ID NO: 439) RE Sites AndNucleotides Within SEQ ID Description Of Fragment And Chimeric Gene NO:439 Components Swa I/BsiW I FBAINm::EgD9ES/EgD8M::Pex20, comprising:(6067-318)  FBAINm: Yarrowia lipolytica FBAINm promoter (WO 2005/049805;U.S. Pat. No. 7,202,356); EgD9ES/EgD8M: gene fusion comprising thecodon-optimized Euglena gracilis delta-9 elongase (EgD9ES), a modifiedlinker derived from EgDHAsyn1, and the synthetic mutant delta-8desaturase derived from Euglena gracilis (EgD8M) (EgD8M is labeled as“EgD8-23” in the Figure) (nucleotide sequence of full-length gene fusionis set forth as SEQ ID NO: 440, while the translated amino acid sequenceis set forth as SEQ ID NO: 441); Pex20: Pex20 terminator sequence ofYarrowia Pex20 gene (GenBank Accession No. AF054613); 1354-474  ColE1plasmid origin of replication 2284-1424 ampicillin-resistance gene(Amp^(R)) for selection in E. coli 3183-4487 Yarrowia autonomousreplication sequence (ARS18; GenBank Accession No. A17608) 6020-4533Yarrowia Ura 3 gene (GenBank Accession No. AJ306421)Generation of Construct pZuFmG9G8fu-B, Comprising a EgD9ES/EgD8M GeneFusion

A BamH I site (i.e., GGATCC) was introduced into pZUFmG9G8fu to generatepZUFmG9G8fu-B (SEQ ID NO:442) using oligonucleotides YL1043 and YL1044(SEQ ID NOs:443 and 444, respectively) as primers and pZuFmG9G8fu astemplate. The BamH I site was located just after the translation startcodon ATG and was in the reading frame of EgD8M, which resulted in a twoamino acid insertion (i.e., glycine and serine) between the methionineamino acid residue and the remaining portion of the EgD8M polypeptide.This modification caused the linker region between EgD9ES and EgD8M tobecome a peptide having the sequence set forth as SEQ ID NO:445 (i.e.,GAGPARPAGLPPATYYDSLAVMGS); the nucleotide and translated amino acidsequence of the full-length EgD9ES/EgD8M gene fusion is set forth as SEQID NOs:446 and 447, respectively.

Generation of Construct pZUFmG9A8, Comprising a EgD9ES/EaD8S Gene Fusion

Plasmid pEaD8S (SEQ ID NO:448) was created when the EaD8S gene (SEQ IDNO:429) was cloned into pUC57 (GenBank Accession No. Y14837). Then, aBamH I site was introduced into pEaD8S using oligonucleotides YL1059 andYL1060 (SEQ ID NOs:449 and 450, respectively) as primers and pEaD8S astemplate, to generate pEaD8S-B (SEQ ID NO:451). The introduced BamH Isite (i.e., GGATCC) was located just before the translation start codonof EaD8S in pEaD8S-B, and is in the same reading frame with EaD8S.

The BamH I/Not I fragment of pEaD8S-B comprising EaD8S was used toreplace the BamH I/Not I fragment of pZUFmG9G8fu-B (SEQ ID NO:442) togenerate pZUFmG9A8 (SEQ ID NO:452; FIG. 55B) (thereby introducing EaD8Sin place of EgD8M). The linker region between EgD9ES and EaD8S was apeptide having the sequence set forth in SEQ ID NO:445 (i.e.,GAGPARPAGLPPATYYDSLAVMGS). Thus, plasmid pZUFmG9A8 contained theEgD9ES/EaD8S gene fusion, flanked by the Yarrowia lipolytica FBAINmpromoter and a Pex20 terminator (GenBank Accession No. AF054613). Thenucleotide and translated amino acid sequence of the full-lengthEgD9ES/EaD8S gene fusion is set forth as SEQ ID NOs:453 and 454,respectively.

Generation of Construct QZUFmA9G8, Comprising a EaD9ES/EgD8M Gene Fusion

Plasmid pZUFmEaD9ES (SEQ ID NO:455) contained the EaD9ES gene, flankedby the Yarrowia lipolytica FBAINm promoter and a Pex20 terminator(GenBank Accession No. AF054613). A Nar I site was introduced into theplasmid to generate pZUFmEaD9ES-Na (SEQ ID NO:456) usingoligonucleotides YL1049 and YL1050 (SEQ ID NOs:457 and 458,respectively) as primers and pZUFmEaD9ES as template. The introduced NarI site (i.e., GGCGCC) was located just before the translation stop codonof EaD9ES, and is in the same reading frame of EaD9ES.

The Nco I/Nar I fragment of pZUFmEaD9ES-Na (SEQ ID NO:456) comprisingEaD9ES was used to replace the Nco I/Nar I fragment of pZUFmG9G8fu-B(SEQ ID NO:442) to generate pZUFmA9G8 (SEQ ID NO:459) (therebyintroducing EaD9ES in place of EgD9ES). The linker region between EaD9ESand EgD8M was a peptide having the sequence set forth in SEQ ID NO:445(i.e., GAGPARPAGLPPATYYDSLAVMGS). Thus, plasmid pZUFmA9G8 contained theEaD9ES/EgD8M gene fusion, flanked by the Yarrowia lipolytica FBAINmpromoter and a Pex20 terminator (GenBank Accession No. AF054613). Thenucleotide and translated amino acid sequence of the full-lengthEaD9ES/EgD8M gene fusion is set forth as SEQ ID NOs:460 and 461,respectively.

Generation of Construct pZUFmA9A8, Comprising a EaD9ES/EaD8S Gene Fusion

The BamH I I/Not I fragment of pEaD8S-B (SEQ ID NO:451) comprising EaD8Swas used to replace the BamH I/Not I fragment of pZUFmA9G8 (SEQ IDNO:459) to generate pZUFmA9A8 (SEQ ID NO:462) (thereby introducing EaD8Sin place of EgD8M). The linker region between EaD9ES and EaD8S was apeptide having the sequence set forth in SEQ ID NO:445 (i.e.,GAGPARPAGLPPATYYDSLAVMGS). Thus, plasmid pZUFmA9A8 contained theEaD9ES/EaD8S gene fusion, flanked by the Yarrowia lipolytica FBAINmpromoter and a Pex20 terminator (GenBank Accession No. AF054613). Thenucleotide and translated amino acid sequence of the full-lengthEaD9ES/EaD8S gene fusion is set forth as SEQ ID NOs:463 and 464,respectively.

Generation of Construct pZUFmR9G8, Comprising a E389D9eS/EgD8M GeneFusion

Plasmid pE389S (SEQ ID NO:465) was created when the E389D9eS gene (SEQID NO:358) was cloned into pUC57 (GenBank Accession No. Y14837). Then, aNarI site was introduced into pE389S to generate pE389S-Na (SEQ IDNO:466) using oligonucleotides YL1051 and YL1052 (SEQ ID NOs:467 and468, respectively) as primers and pE389S as template. The introduced NarI (i.e., GGCGCC) site was located just before the translation stop codonand was in the same reading frame of E389D9eS.

The Nco I/Nar I fragment of pE389S-Na comprising E389D9eS was used toreplace the Nco I/Nar I fragment of pZUFmG9G8fu-B comprising EgD9ES togenerate pZUFmR9G8 (SEQ ID NO:469) (thereby introducing E389D9eS inplace of EgD9ES). The linker region between E389D9eS and EgD8M was apeptide having the sequence set forth in SEQ ID NO:445 (i.e.,GAGPARPAGLPPATYYDSLAVMGS). Thus, plasmid pZUFmR9G8 contained theE389D9eS/EgD8M gene fusion, flanked by the Yarrowia lipolytica FBAINmpromoter and a Pex20 terminator (GenBank Accession No. AF054613). Thenucleotide and translated amino acid sequence of the full-lengthE389D9eS/EgD8M gene fusion is set forth as SEQ ID NOs:470 and 471,respectively.

Example 56 Functional Analyses of Delta-9 Elongase/Delta-8 DesaturaseGene Fusions in Yarrowia lipolytica Strain Y2224

The plasmids from Example 55 [i.e., pZUFmEgD9ES (SEQ ID NO:431),pZUFMEgD9ES-Na (SEQ ID NO:432), pZUFMG9G8fu (SEQ ID NO:439),pZUFmG9G8fu-B (SEQ ID NO:442), pZUFmG9A8 (SEQ ID NO:452), pZUFmA9G8 (SEQID NO:459), pZuFmA9A8 (SEQ ID NO:462) and pZUFmR9G8 (SEQ ID NO:469)]were transformed into Yarrowia lipolytica strain Y2224 individually, asdescribed in the General Methods. The transformants were selected on MMplates. After 2 days growth at 30° C., eight transformants from eachtransformation reaction were streaked out onto new MM plates andincubated for an additional 2 days at 30° C. Once grown, these strainswere individually inoculated into 3 mL liquid MM at 30° C. and shaken at250 rpm/min for 2 days. The cells were collected by centrifugation, thesupernatant was removed and 3 mL of HGM was added. These strains weregrown in a 30° C. incubator shaking at 250 rpm for an additional 5 days.The cells were collected by centrifugation, lipids were extracted, andfatty acid methyl esters were prepared by trans-esterification, andsubsequently analyzed with a Hewlett-Packard 6890 GC.

GC analyses showed that there were both delta-9 elongase and delta-8desaturase activities in all strains having a delta-9 elongase/delta-8desaturase gene fusion. The results are summarized below in Table 34.Delta-9 elongase activity was calculated by dividing the sum of theweight percent (wt %) for EDA and DGLA by the sum of the wt % for LA,EDA and DGLA and multiplying by 100 to express as a percent; similarly,delta-8 desaturase activity was calculated by dividing the wt % for DGLAby the sum of the wt % for EDA and DGLA and multiplying by 100 toexpress as a percent.

TABLE 34 Y2224 Delta-8 Transformant Delta-9 Elongase Desaturase Delta-9Delta-8 (plasmid nucleotide (amino acid (amino acid Linker ElongaseDesaturase SEQ ID NO) SEQ ID NO) SEQ ID NO) (amino acid SEQ ID NO)Activity Activity pZUFmEgD9ES EgD9ES — — 20% — (SEQ ID NO: 431)(SEQ ID NO: 319) pZUFmEgD9ES-Na EgD9ES — — 19% — (SEQ ID NO: 432)(SEQ ID NO: 319) pZUFmG9G8fu EgD9ES EgD8M GAGPARPAGLPPATYYDSLAVM 17% 73%(SEQ ID NO: 439) (SEQ ID NO: 319)  (SEQ ID NO: 328) (SEQ ID NO: 438)pZUFmG9G8fu-B EgD9ES EgD8M GAGPARPAGLPPATYYDSLAVMGS 21% 73%(SEQ ID NO: 442) (SEQ ID NO: 319)  (SEQ ID NO: 328) (SEQ ID NO: 445)pZUFmG9A8 EgD9ES EaD8S GAGPARPAGLPPATYYDSLAVMGS 21% 67% (SEQ ID NO: 452)(SEQ ID NO: 319)  (SEQ ID NO: 430) (SEQ ID NO: 445) pZuFmA9G8 EaD9ESEgD8M GAGPARPAGLPPATYYDSLAVMGS 15% 54% (SEQ ID NO: 459)(SEQ ID NO: 422)  (SEQ ID NO: 328) (SEQ ID NO: 445) pZUFmA9A8 EaD9ESEaD8S GAGPARPAGLPPATYYDSLAVMGS 18% 58% (SEQ ID NO: 462)(SEQ ID NO: 422)  (SEQ ID NO: 430) (SEQ ID NO: 445) pZUFmR9G8 E389D9eSEgD8M GAGPARPAGLPPATYYDSLAVMGS 18% 70% (SEQ ID NO: 469) (SEQ ID NO: 359)(SEQ ID NO: 328) (SEQ ID NO: 445)

Delta-9 Elongase and Delta-8 Desaturase Activities in YarrowiaTransformed with Various Gene Fusions

In summary of Table 34, the data showed that all six fusion genes hadboth delta-9 elongase and delta-8 desaturase activities; thus, thefusion proteins from the fusion genes effectively permitted expressionof two independent and separable enzymatic activities. More importantly,fusing the two independent enzymes together as one fusion proteinseparated by a linker region increased flux from LA to DGLA. In allcases, the fusion gene had higher activity than at least one of theindividual genes when expressed alone in Yarrowia. These data suggestedthat the product of delta-9 elongase may be directly channeled assubstrate of delta-8 desaturase in the fusion protein.

More specifically, in the case of the EgD9ES/EgD8M gene fusion (i.e.,pZuFmG9G8fu-B) and the EgD9ES/EaD8S gene fusion (i.e., pZuFmG9A8), theEgD9ES delta-9 elongase (21% conversion) performed in a mannercomparable to that when EgD9ES was expressed alone (20% conversion[pZuFmEgD9ES data and Example 55]). In contrast, however, the EgD8Mdelta-8 desaturase activity in Yarrowia expressing pZuFmG9G8-B was about97% more efficient than when EgD8M was expressed alone (73% versus 37%conversion [Example 55]). Similarly, the EaD8S delta-8 desaturaseactivity in Yarrowia expressing pZuFmG9A8 was about 63% more efficientthan when EaD8S was expressed alone (67% versus 41% conversion [Example55]).

In the case of the EaD9ES/EgD8M gene fusion (i.e., pZuFmA9G8) and theEaD9ES/EaD8S gene fusion (i.e., pZuFmA9A8), the EaD9ES delta-9 elongaseactivity was about 15% and 38% more efficient, respectively, than whenEaD9ES was expressed alone (15% and 18% conversion, respectively, versus13% conversion [Example 55]). Likewise, the EgD8M delta-8 desaturaseactivity in Yarrowia expressing pZuFmA9G8 was about 46% more efficientthan when EgD8M was expressed alone (54% versus 37% conversion [Example55]). Similarly, the EaD8S delta-8 desaturase activity in Yarrowiaexpressing pZuFmA9A8 was about 32% more efficient than when EaD8S wasexpressed alone (58% versus 41% conversion [Example 55]).

Finally, in the case of the E389D9eS/EgD8M gene fusion (i.e.,pZuFmR9G8), the E389D9eS delta-9 elongase activity was about 50% moreefficient than when E389D9eS was expressed alone (18% versus 12%conversion [Example 55]). Likewise, the EgD8M delta-8 desaturaseactivity in Yarrowia expressing pZuFmR9G8 was about 89% more efficientthan when EgD8M was expressed alone (70% versus 37% conversion [Example55]).

Table 34 also demonstrated that the modified linkerGAGPARPAGLPPATYYDSLAVMGS (SEQ ID NO:445) was preferred as opposed to thelinker set forth as SEQ ID NO:438 in Yarrowia lipolytica, when fusingdelta-9 elongase and delta-8 desaturase genes together.

It will be obvious to one of skill in the art that other PUFA desaturaseand elongase genes that are preferred for expression in Yarrowialipolytica (including, for example, any of those genes described inTables 8-19) can be fused together in a manner similar to that describedabove and expressed in Yarrowia lipolytica. Preferred promoters andterminators suitable for construction of an expression cassette (whereinthe ORF expressed encodes a multizyme) may be selected from thosedescribed in Tables 8-19. It is hypothesized that increased efficiencyor flux would be observed in the fusion gene as opposed to when either(or both) individual genes are expressed alone.

Example 57 Creation of Delta-9 Elongase/Delta-8 Desaturase Gene Fusionsfor Expression in Soy

In order to characterize multizyme fusions between delta-9 elongases anddelta-8 desaturases in soy, a series of delta-9 elongase/delta-8multizymes were created. Delta-9 elongase and delta-8 desaturase domainswere separated by the EgDHAsyn1 proline-rich linker (SEQ ID NO:198). Forcomparison, constructs that co-expressed individual delta-9 elongase anddelta-8 desaturase genes were also created. Delta-9 elongases usedinclude EgD9elo (Example 16; SEQ ID NO:112; also referred to as EgD9eand EgD9E herein, but they are identical) and EaD9elo1 (SEQ ID NO:252;Example 36; also referred to as EaD9E and EaD9e herein, but they are allidentical). Delta-8 desaturases used include TpomD8 (SEQ ID NO:162;Example 21) and the Euglena anabaena delta-8 desaturase (EaD8Des3; SEQID NO:427; also referred to as EaD8 but they are identical; described inU.S. Provisional Application No. 60/910,831(filed Apr. 10, 2007;Attorney Docket No. BB-1615).

In the present Example and for Example 23, which describes the synthesisof the EgD9elo-EgDHAsyn1Link-PavD8 fusion, additional nucleotides wereadded to the 3′ end of the EgDHAsyn1 proline-rich linker sequence toenable cloning when making the fusions for all constructs. Thus, anadditional 4 amino acids were included between the end of the EgDHAsyn1proline-rich linker (SEQ ID NO:198; PARPAGLPPATYYDSLAV) and the start ofthe delta-8 desaturase used (i.e. SEQ ID NO:472;PARPAGLPPATYYDSLAVSGRT).

Plasmid pKR1183 (SEQ ID NO:266) comprising the Euglena anabaena delta-9elongase-Tetruetreptia pomquetensis CCMP1491 delta-8 desaturase fusion(Hybrid1-HGLA Synthase; also called EaD9e/TpomD8) was described inExample 38.

Other plasmids described in the present example include: pKR1014(described in U.S. patent application Ser. No. 11/876,115 (filed Oct.22, 2007; Attorney Docket No. BB-1574; comprising EgD9e and TpomD8expressed individually), pKR1152 (comprising an EgD9e and EaD8 expressedindividually), pKR1151 (comprising an EaD9e and TpomD8 expressedindividually), pKR1150 (comprising an EaD9e and EaD8 expressedindividually), pKR1184 (comprising a EaD9e/EaD8 gene fusion), pKR1199(comprising a EgD9e/TpomD8 gene fusion), and pKR1200 (comprising aEgD9e/EaD8 gene fusion). A summary of the constructs made and respectivegenes tested along with the SEQ ID NOs for the nucleotide and amino acidsequences produced is shown in Table 35.

Functional analysis of the activity of each gene fusion is tested infra,in Example 60.

TABLE 35 Preferred Desaturases And Elongases For Creation OfDelta-9/Delta-8 Gene Fusions In Soy SEQ ID NOs for SEQ ID NOs for VectorSEQ ID Gene(s) nucleotide amino acid Vector NOs Expressed sequence(s)sequences pKR1014 SEQ ID NO: 474 EgD9e SEQ ID NO: 112 SEQ ID NO: 513TpomD8 SEQ ID NO: 162 SEQ ID NO: 514 pKR1152 SEQ ID NO: 479 EgD9e SEQ IDNO: 112 SEQ ID NO: 513 EaD8 SEQ ID NO: 427 SEQ ID NO: 428 pKR1151 SEQ IDNO: 484 EaD9e SEQ ID NO: 252 SEQ ID NO: 254 TpomD8 SEQ ID NO: 162 SEQ IDNO: 514 pKR1150 SEQ ID NO: 485 EaD9e SEQ ID NO: 252 SEQ ID NO: 254 EaD8SEQ ID NO: 427 SEQ ID NO: 428 pKR1199 SEQ ID NO: 488 EgD9e/TpomD8 SEQ IDNO: 492 SEQ ID NO: 515 fusion pKR1200 SEQ ID NO: 490 EgD9e/EaD8 SEQ IDNO: 493 SEQ ID NO: 516 fusion pKR1183 SEQ ID NO: 266 EaD9e/TpomD8 SEQ IDNO: 494 SEQ ID NO: 517 fusion pKR1184 SEQ ID NO: 491 EaD9e/EaD8 SEQ IDNO: 495 SEQ ID NO: 518 fusion KS373 SEQ ID NO: 179 EgD9e/PavD8 SEQ IDNO: 496 SEQ ID NO: 519 fusion

Construction of pKR1014

Vector pKR123r, which was previously described in PCT Publication No. WO2004/071467 (published Aug. 26, 2004), contains a NotI site flanked bythe Kunitz soybean Trypsin Inhibitor (KTi3) promoter (Jofuku et al.,Plant Cell 1:1079-1093 (1989)) and the KTi 3′ termination region, theisolation of which is described in U.S. Pat. No. 6,372,965(KTi3/NotI/KTi3′ cassette). TpomD8 (SEQ ID NO:162; Example 21) wasreleased from pLF114-10 (SEQ ID NO:165; Example 21) by digestion withNotI and cloned into the NotI site of pKR123r to produce pKR1007 (SEQ IDNO:473).

Vector pKR912, which was previously described in US-2007-0118929-A1 andpublished May 24, 2007, contains the hygromycin B phosphotransferasegene, flanked by the 35S promoter (Odell et al., Nature 313:810-812(1985)) and NOS 3′ transcription terminator (Depicker et al., J. Mol.Appl. Genet. 1:561-570 (1982)) (35S/hpt/NOS3′ cassette) for selection inplants such as soybean. Vector pKR912 also contains EgD9e (SEQ IDNO:112), flanked by the promoter for the α′ subunit of β-conglycinin(Beachy et al., EMBO J. 4:3047-3053 (1985)) and the 3′ transcriptiontermination region of the phaseolin gene (Doyle et al., J. Biol. Chem.261:9228-9238 (1986)), thus allowing for strong tissue-specificexpression of EgD9e in the seeds of soybean.

Plasmid pKR1007 (SEQ ID NO:473) was digested with PstI, and the fragmentcontaining the Tetruetreptia pomquetensis delta-8 desaturase was clonedinto the SbfI site of pKR912, to give pKR1014 (SEQ ID NO:474). In thisway, the Tetruetreptia pomquetensis delta-8 desaturase is co-expressedwith the Euglena gracilis delta-9 elongase behind strong, seed-specificpromoters. A schematic depiction of pKR1014 is shown in FIG. 56. In FIG.56, TpomD8 is called Tetruetreptia pomquetensis 1491 delta-8 Desaturase,and EgD9e is called eug el1.

Construction of pKR1151

In order to introduce NotI and NcoI restriction sites at the 5′ end ofthe coding sequences and a NotI site at the 3′ end of the codingsequences, EaD8 (SEQ ID NO:427) was amplified with oligonucleotideprimers EaD8-5 (SEQ ID NO:475) and EaD8-3 (SEQ ID NO:476) using thePhusion™ High-Fidelity DNA Polymerase (Cat. No. F553S, Finnzymes Oy,Finland) following the manufacturer's protocol. The resulting DNAfragment was cloned into the pCR-Blunt® cloning vector using the ZeroBlunt® PCR Cloning Kit (Invitrogen Corporation), following themanufacturer's protocol, to produce pLF120-3 (SEQ ID NO:477).

Vector pLF120-3 (SEQ ID NO:477), was digested with NotI, and thefragment containing EaD8 was cloned into the NotI site of pKR457 (SEQ IDNO:122; Example 16), to produce pKR1138 (SEQ ID NO:478).

Vector pKR1138 (SEQ ID NO:478) was digested with BsiWI, and the fragmentcontaining EaD8 was cloned into the BsiWI site of pKR912 to give pKR1152(SEQ ID NO:479). A schematic depiction of pKR1152 is shown in FIG. 57.In FIG. 57, EaD8 is called EaD8Des3, and EgD9e is called eug el1 .

Construction of pKR1152

In order to introduce NotI and NcoI restriction sites at the 5′ end ofthe coding sequences and a NotI site at the 3′ end of the codingsequences, EaD9e was PCR amplified from pLF121-1 (SEQ ID NO:250; Example36) with oligonucleotide primers oEAd9el1-1 (SEQ ID NO:298; Example 44)and oEAd9el1-2 (SEQ ID NO:480) using the Phusion™ High-Fidelity DNAPolymerase (Cat. No. F553S, Finnzymes Oy, Finland) following themanufacturer's protocol. The resulting DNA fragments were cloned intothe pCR-Blunt cloning vector using the Zero Blunt PCR Cloning Kit(Invitrogen Corporation), following the manufacturer's protocol, toproduce pKR1137 (SEQ ID NO:481).

EaD9e was released from pKR1137 (SEQ ID NO:481) by digestion with NotIand cloned into the NotI site of pKR72 (SEQ ID NO:105; Example 15) toproduce pKR1140 (SEQ ID NO:482).

TpomD8 was released from pLF114-10 (SEQ ID NO:165; Example 21) bydigestion with NotI and was cloned into the NotI site of plasmid pKR457(SEQ ID NO:122; Example 16) to produce pKR1145 (SEQ ID NO:483).

Vector pKR1145 (SEQ ID NO:483) was digested with BsiWI and the fragmentcontaining TpomD8 was cloned into the BsiWI site of pKR1140 (SEQ IDNO:482) to give pKR1151 (SEQ ID NO:484). A schematic depiction ofpKR1151 is shown in FIG. 58. In FIG. 58, TpomD8 is called Tetruetreptiapomquetensis 1491 delta-8 Desaturase, and EaD9e is called EAd9elong.

Construction of pKR1150

Vector pKR1138 (SEQ ID NO:478) was digested with BsiWI, and the fragmentcontaining EaD8 was cloned into the BsiWI site of pKR1140 (SEQ IDNO:482) to give pKR1150 (SEQ ID NO:485). A schematic depiction ofpKR1150 is shown in FIG. 59. In FIG. 59, EaD8 is called EaD8Des3, andEaD9e is called EAd9elong.

Construction of pKR1199

The NcoI/NotI DNA fragment of KS373 (SEQ ID NO:179; Example 23),containing EgD9elo-EgDHAsyn1Link, was cloned into the NcoI/NotI DNAfragment from pKR1177 (SEQ ID NO:264; Example 38), containing thepromoter for the α′ subunit of β-conglycinin, to produce pKR1190 (SEQ IDNO:486).

The NotI fragment from pLF114-10 (SEQ ID NO:165; Example 21), containingTpomD8, was cloned into the NotI fragment of pKR1190 (SEQ ID NO:486) toproduce pKR1195 (SEQ ID NO:487).

The BamHI DNA fragment of pKR1195 (SEQ ID NO:487), containing theEgD9e/TpomD8 fusion gene, was cloned into the BamHI DNA fragment ofpKR325, previously described in PCT Publication No. WO 2006/012325 toproduce pKR1199 (SEQ ID NO:488). A schematic depiction of pKR1199 isshown in FIG. 60. In FIG. 60, EgD9e/TpomD8 is called EGd9elong-TPOMd8DS.

Construction of pKR1200

The NotI fragment from pLF120-3 (SEQ ID NO:477), containing EaD8 wascloned into the NotI fragment of pKR1190 (SEQ ID NO:486) to producepKR1196 (SEQ ID NO:489).

The BamHI DNA fragment of pKR1196 (SEQ ID NO:489), containing theEgD9e/EaD8 fusion gene, was cloned into the BamHI DNA fragment ofpKR325, previously described in PCT Publication No. WO 2006/012325 toproduce pKR1200 (SEQ ID NO:490). A schematic depiction of pKR1200 isshown in FIG. 61. In FIG. 61, EgD9e/EaD8 is called EGd9ELONG-EAd8DS.

Construction of pKR1184

The NotI fragment from pLF120-3 (SEQ ID NO:477), containing EaD8, wascloned into the NotI fragment of pKR1179 (SEQ ID NO:265) to producepKR1184 (SEQ ID NO:491). A schematic depiction of pKR1184 is shown inFIG. 62. In FIG. 62, EaD9e/EaD8 is called EAd9ELONG-EAd8DS.

Example 58 Construction of Soybean Expression Vector pKR1321 forExpression of a Tetruetreptia pomquetensis CCMP1491 Delta-8Desaturase-Euglena anabaena delta-9 elongase Fusion Gene(TpomD8-EaD9Elo1 fusion)

The present example describes the construction of an in-frame fusiongene between the Tetruetreptia pomquetensis CCMP1491 delta-8 Desaturase(TpomD8; SEQ ID NO:162; Example 21) and the Euglena anabaena delta-9elongase (EaD9e; SEQ ID NO:252, Example 36). Each domain is separated bythe EgDHAsyn1 linker with an additional 4 amino acids included betweenthe end of the EgDHAsyn1 proline-rich linker and the start of the EaD9e,as described in Example 57 (i.e. SEQ ID NO:472; PARPAGLPPATYYDSLAVSGRT).

Plasmid pKR1301 (SEQ ID NO:307; Example 44) was digested with EcoRI, andthe DNA fragment containing the 3′ end of TpomD8-EgDHAsyn1Link (calledTpomD8+L1TR1) was re-ligated to form pKR1303 (SEQ ID NO:497).

The NotI fragment of pKR1137 (SEQ ID NO:481; Example 57), containing theEaD9e, was cloned into the EagI site of pKR1303 (SEQ ID NO:497) toproduce pKR1308 (SEQ ID NO:498). In this way, EaD9e was fused to the 3′end of TpomD8.

The Gy1/Pavelo/legA2 cassette was released from plasmid pKR336(described in PCT Publication Nos. WO 04/071467; the contents of whichare hereby incorporated by reference) by digestion with PstI/BamHI andcloned into the PstI/BamHI site of pKR268 (described in PCT PublicationNos. WO 04/071467) to produce pKR393 (SEQ ID NO:499). The Pavelo genewas released from pKR393 (SEQ ID NO:499) by digestion with NotI, and thevector was re-ligated to form pKR407 (SEQ ID NO:500).

TpomD8 was released from pLF114-10 (SEQ ID NO:165; Example 21) bydigestion with NotI and was cloned into the NotI site of plasmid pKR407(SEQ ID NO:500) to produce pKR1018 (SEQ ID NO:501).

Plasmid pKR1018 (SEQ ID NO:501) was digested with HindIII/EcoRI, and thefragment containing the 5′ end of the Tpomd8 was cloned into theHindIII/EcoRI site of pKR1308 (SEQ ID NO:498) to produce pKR1312 (SEQ IDNO:502). In this way, the TpomD8 sequence was restored, and theTpomD8/EaD9e fusion was formed.

The NotI fragment of pKR1312 (SEQ ID NO:502), containing theTpomD8/EaD9e fusion, was cloned into the NotI site of pKR72 (SEQ IDNO:105; Example 23) to produce pKR1321 (SEQ ID NO:503). A schematicdepiction of pKR1321 is shown in FIG. 63. In FIG. 63, TpomD8/EaD9e iscalled TPd8ds-EAd9el fusion.

Example 59 Construction of Soybean Expression Vector pKR1326 forExpression of a Euglena anabaena Delta-9 Elongase-Tetruetreptiapomquetensis CCMP1491 Delta-8 Desaturase Fusion Gene Using the Euglenaanabaena DHAsyn1 Proline-Rich Linker

The present example describes the construction of an in-frame fusiongene between the Euglena anabaena delta-9 elongase (EaD9e; SEQ IDNO:252, Example 36) and the Tetruetreptia pomquetensis CCMP1491 delta-8Desaturase (TpomD8; SEQ ID NO:162; Example 21). Each domain is separatedby the EaDHAsyn1 proline-rich linker (SEQ ID NO:235), but with anadditional 3 amino acids included between the end of the EaDHAsyn1proline-rich linker (EaDHAsyn1 Link) and the start of the EaD9e (i.e.SEQ ID NO:504; PGGPGKPSEIASLPPPIRPVGNPPAAYYDALATGRT). Cloning wasperformed as similarly described in Example 57.

An initial in-frame fusion between the EaD9e and the EaDHAsyn1 Link(EaD9elo-EgDHAsyn1Link) was made by PCR amplification and was flanked bya NotI and NcoI site at the 5′ end and a NotI site at the 3′ end. EaD9e(SEQ ID NO:252) was amplified from pLF121-1 (SEQ ID NO:250) witholigonucleotides oEAd9el1-1 (SEQ ID NO:298) and EaLink1 (SEQ ID NO:505),using the Phusion™ High-Fidelity DNA Polymerase (Cat. No. F553S,Finnzymes Oy, Finland) following the manufacturer's protocol. EaDHAsyn1Link (SEQ ID NO:234) was amplified in a similar way from pLF117-1 (SEQID NO:87; Example 13) with oligonucleotides EaLink2 (SEQ ID NO:506) andEaLink3 (SEQ ID NO:507). The two resulting PCR products were combinedand re-amplified using oEAd9el1-1 (SEQ ID NO:298) and EaLink3 (SEQ IDNO:507) to form EaD9e-EaDHAsyn1 Link. The sequence of theEaD9e-EaDHAsyn1 Link is shown in SEQ ID NO:508. EaD9e-EaDHAsyn1 Link wascloned into the pCR-Blunt® cloning vector using the Zero Blunt PCRCloning Kit (Invitrogen Corporation), following the manufacturer'sprotocol, to produce pKR1305 (SEQ ID NO:509).

The EagI DNA fragment of pKR1305 (SEQ ID NO:509), containingEaD9e-EaDHAsyn1Link, was cloned into the NotI site pKR1304 (SEQ IDNO:310; Example 44) to produce pKR1317 (SEQ ID NO:510). In this way, the5′ end of the TpomD8 was fused to EaD9e-EaDHAsyn1 Link.

The EcoRI/Asp718 fragment of pKR1127 (SEQ ID NO:168; Example 22),containing the 3′ end of the TpomD8 was cloned into the EcoRI/Asp718fragment of pKR1317 (SEQ ID NO:510), containing EaD9e-EaDHAsyn1Link toproduce pKR1320 (SEQ ID NO:511).

The NotI fragment from pKR1320 (SEQ ID NO:511), containing the fusion,was cloned into the NotI fragment of pKR72 (SEQ ID NO:105; Example 15)to produce pKR1326 (SEQ ID NO:512). A schematic depiction of pKR1326 isshown in FIG. 64. In FIG. 64, EaD9e/TpomD8 with the EaDHAsyn1proline-rich linker is called EAd9el-TPOMd8ds L2fusion.

Example 60 Functional Analyses of Delta-9 Elongase/Delta-8 DesaturaseGene Fusions in Soy

The present example describes the transformation and expression insoybean somatic embryos of pKR1014 (SEQ ID NO:474), pKR1152 (SEQ IDNO:479), pKR1151 (SEQ ID NO:484), pKR1150 (SEQ ID NO:485), pKR1199 (SEQID NO:488), pKR1200 (SEQ ID NO:490), and pKR1184 (SEQ ID NO:491), thesyntheses of which were previously described in Example 57. Functionalanalyses of pKR1183 (SEQ ID NO:266) and KS373 (SEQ ID NO:179) werepreviously described in Examples 46 and 31, respectively.

Soybean embryogenic suspension culture (cv. Jack) was transformed witheach of the vectors above, and embryos were matured in soybeanhistodifferentiation and maturation liquid medium (SHaM liquid media;Schmidt et al., Cell Biology and Morphogenesis, 24:393 (2005)), asdescribed in Example 25 and previously described in PCT Publication No.WO 2007/136877, published Nov. 29, 2007 (the contents of which arehereby incorporated by reference).

After maturation in SHaM liquid media, a subset of transformed soybeanembryos (i.e., 5-6 embryos per event) were harvested and analyzed asdescribed herein.

In this way, approximately 30 events transformed with pKR1014 (SEQ IDNO:474), pKR1152 (SEQ ID NO:479), pKR1151 (SEQ ID NO:484), pKR1150 (SEQID NO:485), pKR1199 (SEQ ID NO:488), pKR1200 (SEQ ID NO:490), or pKR1184(SEQ ID NO:491) were analyzed. The five events having the highestaverage DGLA content (average of the 5 embryos analyzed) are shown inFIG. 65, 66, 67, 68, 69, 70, or 71, respectively. In FIGS. 65-71, fattyacids are identified as 16:0 (palmitate), 18:0 (stearic acid), 18:1(oleic acid), LA, ALA, EDA, ERA, DGLA, and ETA. Fatty acid compositionsare expressed as a weight percent (wt. %) of total fatty acids. Table 36summarizes the vector, genes used, experiment number (MSE#), andcorresponding FIG.

In FIGS. 65-71, elongation activity is expressed as % delta-9 elongationof C18 fatty acids (C18% delta-9 elong), calculated according to thefollowing formula: ([product]/[substrate+product])*100. Morespecifically, the combined percent elongation for LA and ALA isdetermined as: ([DGLA+ETA+EDA+ERA]/[LA+ALA+DGLA+ETA+EDA+ERA])*100.

In FIGS. 65-71, the combined percent desaturation for EDA and ERA isshown as “C20% delta-8 desat”, determined as:([DGLA+ETA]/[DGLA+ETA+EDA+ERA])*100. This is also referred to as theoverall % desaturation.

TABLE 36 Functional analysis of Delta-9/Delta-8 Gene Fusions In Soy MSEFIG. # Containing Vector Gene(s) Expressed Experiment FunctionalAnalysis pKR1014 EgD9e MSE2024 FIG. 65 TpomD8 pKR1152 EgD9e MSE2136 FIG.66 EaD8 pKR1151 EaD9e MSE2131 FIG. 67 TpomD8 pKR1150 EaD9e MSE2130 FIG.68 EaD8 pKR1199 EgD9e/TpomD8 MSE2153 FIG. 69 fusion pKR1200 EgD9e/EaD8fusion MSE2154 FIG. 70 pKR1183 EaD9e/TpomD8 fusion MSE2145 FIG. 37(Example 46)* pKR1184 EaD9e/EaD8 fusion MSE2146 FIG. 71 KS373EgD9e/PavD8 fusion MSE2071 FIG. 24 (Example 31) *In FIG. 37, MSE2145 islisted as MSE2144

A comparison of individually expressed delta-9 elongases with delta-8desaturases versus the equivalent delta-9 elongase-delta-8 desaturasefusion is shown in FIG. 72. In FIG. 72, each data point represents theaverage % DGLA or % EDA for 5-6 embryos (as a % of total fatty acids)for all events analyzed, and avg. % DGLA is plotted vs. avg. % EDA. InFIG. 72A, EgTpom represents EgD9e co-expressed with TpomD8 (pKR1014),and EgTpomfus represents the EgD9e/TpomD8 fusion (pKR1199). In FIG. 72B,EgEa represents EgD9e co-expressed with EaD8 (pKR1152), and EgEafusrepresents the EgD9e/EaD8 fusion (pKR1200). In FIG. 72C, EaTpomrepresents EaD9e co-expressed with TpomD8 (pKR1151), and EaTpomfusrepresents the EaD9e/TpomD8 fusion (pKR1183). In FIG. 72D, EaEarepresents EaD9e co-expressed with EaD8 (pKR1150), and EaEafusrepresents the EaD9e/EaD8 fusion (pKR1200).

Example 61 Functional Analyses of Delta-9 Elongase/Delta-8Desaturase/Delta-5 Desaturase Gene Fusion

The present example describes the transformation and expression insoybean somatic embryos of pKR1322 (SEQ ID NO:314; Example 50)comprising a EaD9Elo1-TpomD8-EaD5Des1 triple fusion (also calledEaD9e/TpomD8/EaD5). Each domain is separated by the EgDHAsyn1 linkerwith an additional 4 amino acids (i.e. SEQ ID NO:472;PARPAGLPPATYYDSLAVSGRT).

Soybean embryogenic suspension culture (cv. Jack) was transformed withpKR1322, and embryos were matured in soybean histodifferentiation andmaturation liquid medium (SHaM liquid media; Schmidt et al., CellBiology and Morphogenesis, 24:393 (2005)) as described in Example 25 andpreviously described in PCT Publication No. WO 2007/136877, publishedNov. 29, 2007 (the contents of which are hereby incorporated byreference).

After maturation in SHaM liquid media, a subset of transformed soybeanembryos (i.e., 5embryos per event) were harvested and analyzed asdescribed herein.

In this way, approximately 30 events transformed with pKR1322(Experiment MSE2274) were analyzed, and the five events having thehighest average ARA and EPA content (average of the 5 embryos analyzed)are shown in FIG. 73. In FIG. 73, fatty acids are identified as 16:0(palmitate), 18:0 (stearic acid), 18:1 (oleic acid), 18:2 (5,9), LA,ALA, EDA, ERA, SCI, DGLA, JUN (also called JUP), ETA, ARA, and EPA.Fatty acid compositions are expressed as a weight percent (wt. %) oftotal fatty acids.

In FIG. 73, elongation activity is expressed as % delta-9 elongation ofC18 fatty acids (% Elo), calculated according to the following formula:([product]/[substrate+product])*100. More specifically, the combinedpercent elongation for LA and ALA is determined as:([DGLA+ETA+EDA+ERA+EPA+ARA]/[LA+ALA+DGLA+ETA+EDA+ERA+EPA+ARA])*100. InFIG. 73, the combined percent delta-8 desaturation for EDA and ERA isshown as “% D8”, determined as:([DGLA+ETA+EPA+ARA]/[DGLA+ETA+EDA+ERA+EPA+ARA])*100. This is alsoreferred to as the overall % delta-8 desaturation.

In FIG. 73, the combined percent delta-5 desaturation for DGLA and ETAis shown as “% D5”, determined as: ([EPA+ARA]/[DGLA+ETA+EPA+ARA])*100.This is also referred to as the overall % delta-5 desaturation.

In summary of FIG. 73, all three domains are functional. This fusioncould be referred to as either EPA synthase or ARA synthase.

Example 62 Functional Analyses of Delta-9 Elongase/Delta-8 Desaturaseand Delta-8 Desaturase/Delta-9 Elongase Gene Fusions

The present example describes the transformation and expression insoybean somatic embryos of either pKR1326 (SEQ ID NO:512), comprising anEaD9Elo1-TpomD8 fusion (also called EaD9e/TpomD8) separated by theEaDHAsyn1 linker with an additional 3 amino acids (i.e. SEQ ID NO:504;PGGPGKPSEIASLPPPIRPVGNPPAAYYDALATGRT), or pKR1321 (SEQ ID NO:503;Example 58), comprising TpomD8/EaD9e fusion separated by the EgDHAsyn1linker.

Soybean embryogenic suspension culture (cv. Jack) was transformed withpKR1326 or pKR1321, and embryos were matured in soybeanhistodifferentiation and maturation liquid medium (SHaM liquid media;Schmidt et al., Cell Biology and Morphogenesis, 24:393 (2005)), asdescribed in Example 25 and previously described in PCT Publication No.WO 2007/136877, published Nov. 29, 2007 (the contents of which arehereby incorporated by reference).

After maturation in SHaM liquid media, a subset of transformed soybeanembryos (i.e., 5embryos per event) were harvested and analyzed asdescribed herein.

In this way, approximately 30 events transformed with pKR1326(Experiment MSE2275) were analyzed, and the five events having thehighest average DGLA and ETA content (average of the 5 embryos analyzed)are shown in FIG. 74. In FIG. 74, fatty acids are identified as 16:0(palmitate), 18:0 (stearic acid), 18:1 (oleic acid), LA, ALA, EDA, ERA,DGLA and DGLA, and ETA. Fatty acid compositions are expressed as aweight percent (wt. %) of total fatty acids.

In FIG. 74, elongation activity is expressed as % delta-9 elongation ofC18 fatty acids (C18% delta-9 elong), calculated according to thefollowing formula: ([product]/[substrate+product])*100. Morespecifically, the combined percent elongation for LA and ALA isdetermined as: ([DGLA+ETA+EDA+ERA]/[LA+ALA+DGLA+ETA+EDA+ERA])*100.

In FIG. 74, the combined percent desaturation for EDA and ERA is shownas “C20% delta-8 desat”, determined as:([DGLA+ETA]/[DGLA+ETA+EDA+ERA])*100. This is also referred to as theoverall % desaturation.

In summary of FIG. 74, the EaDHAsyn1 linker functions similarly to theEgDHAsyn1 linker. No activity was detected for any of the eventstransformed with pKR1321 where TpomD8 was fused to EaD9e with theEgDHAsyn1 linker.

1. A multizyme comprising a single polypeptide having at least twoindependent and separable enzymatic activities.
 2. (canceled)
 3. Themultizyme of claim 1, wherein the enzymatic activities comprises atleast one fatty acid elongase linked to at least one fatty aciddesaturase.
 4. The multizyme of claim 3, wherein the fatty aciddesaturase is selected from the group consisting of a delta-4desaturase, a delta-5 desaturase, a delta-6 desaturase, a delta-8desaturase, a delta-9 desaturase, a delta-12 desaturase, a delta-15desaturase, and a delta-17 desaturase.
 5. The multizyme of claim 3,wherein the fatty acid elongase is selected from the group consisting ofa delta-9 elongase, a C_(14/16) elongase, a C_(16/18) elongase, aC_(18/20) elongase, and a C_(20/22) elongase.
 6. The multizyme of claim1, wherein a first enzymatic activity is linked to a second enzymaticactivity and said link is selected from the group consisting of apolypeptide bond, SEQ ID NO:198 (EgDHAsyn1 linker), SEQ ID NO:200(EgDHAsyn2 linker), SEQ ID NO:235 (EaDHAsyn1 linker), SEQ ID NO:438, SEQID NO:472, SEQ ID NO:445, and SEQ ID NO:504.
 7. An isolatedpolynucleotide encoding a DHA synthase comprising: (a) a nucleotidesequence encoding a polypeptide having DHA synthase activity, whereinthe polypeptide has at least 80% amino acid identity, based on theClustal V method of alignment, when compared to an amino acid sequenceas set forth in SEQ ID NO:12, SEQ ID NO:22, SEQ ID NO:95, SEQ ID NO:96,or SEQ ID NO:97; (b) a nucleotide sequence encoding a polypeptide havingDHA synthase activity wherein the nucleotide sequence has at least 80%sequence identity, based on the BLASTN method of alignment, whencompared to a nucleotide sequence as set forth in SEQ ID NO:11, SEQ IDNO:205, SEQ ID NO:21, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, or SEQID NO:410; (c) a nucleotide sequence encoding a polypeptide having DHAsynthase activity, wherein the nucleotide sequence hybridizes understringent conditions to a nucleotide sequence as set forth in SEQ IDNO:11, SEQ ID NO:205, SEQ ID NO:21, SEQ ID NO:91, SEQ ID NO:92, SEQ IDNO:93, or SEQ ID NO:410; or (d) a complement of the nucleotide sequenceof (a), (b), or (c), wherein the complement and the nucleotide sequenceconsist of the same number of nucleotides and are 100% complementary. 8.The polynucleotide of claim 7, wherein the nucleotide sequence comprisesSEQ ID NO:11, SEQ ID NO:21, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, orSEQ ID NO:
 410. 9. The polynucleotide of claim 7, wherein the amino acidsequence of the polypeptide comprises SEQ ID NO:12, SEQ ID NO:22, SEQ IDNO:95, SEQ ID NO:96, or SEQ ID NO:97. 10-14. (canceled)
 15. Arecombinant construct comprising the isolated polynucleotide of claim 7operably linked to at least one regulatory sequence.
 16. A host cellcomprising in its genome the recombinant construct of claim
 15. 17. Thehost cell of claim 16, wherein said cell is selected from the groupconsisting of plants and yeast.
 18. The host cell of claim 17, whereinthe host cell is a transformed Yarrowia sp. 19-22. (canceled)
 23. Aplant comprising in its genome the recombinant construct of claim 15.24. The plant of claim 23, wherein the plant is an oilseed plant. 25.The plant of claim 23, wherein the plant is soybean.
 26. Seed obtainedfrom the plant of claim
 23. 27-36. (canceled)
 37. A method for making amultizyme which comprises: (a) linking a first polypeptide with at leasta second polypeptide wherein each polypeptide has an independent andseparable enzymatic activity; and (b) evaluating the product of step (a)for the independent and separable enzymatic activities.
 38. (canceled)39. The method of claim 37, wherein the enzymatic activities comprisesat least one fatty acid elongase linked to at least one fatty aciddesaturase.
 40. The method of claim 39, wherein the fatty aciddesaturase is selected from the group consisting of a delta-4desaturase, a delta-5 desaturase, a delta-6 desaturase, a delta-8desaturase, a delta-9 desaturase, a delta-12 desaturase, a delta-15desaturase, and a delta-17 desaturase.
 41. The method of claim 39,wherein the fatty acid elongase is selected from the group consisting ofa delta-9 elongase, a C_(14/16) elongase, a C_(16/18) elongase, aC_(18/20) elongase, and a C_(20/22) elongase.
 42. The method of claim37, wherein the link is selected from the group consisting of apolypeptide bond, SEQ ID NO:198 (EgDHAsyn1 linker amino acid sequence),SEQ ID NO:200 (EgDHAsyn2 linker), SEQ ID NO:235 (EaDHAsyn1 linker), SEQID NO:435, SEQ ID NO:438, SEQ ID NO:472, and SEQ ID NO:504. 43-50.(canceled)
 51. A method for the conversion of eicosapentaenoic acid todocosahexaenoic acid comprising: a) providing a recombinant microbialhost cell comprising: i) a DHA synthase comprising: 1) at least onepolypeptide encoding a C20 elongase; 2) at least one polypeptideencoding a delta-4 desaturase; and 3) a polypeptide linker wherein thelinker is interposed between the C20 elongase and the delta-4desaturase; and ii) a source of eicosapentaenoic acid; and b) growingthe host cell of (a) under conditions whereby docosahexaenoic acid isproduced. 52-65. (canceled)